Genetic architecture of resistance to spot blotch in wheat was examined using a Genome-Wide Association Study (GWAS) involving an association panel comprising 303 diverse genotypes. The association panel was evaluated at two different locations in India including Banaras Hindu University (BHU), Varanasi (Uttar Pradesh), and Borlaug Institute for South Asia (BISA), Pusa, Samastipur (Bihar) for two consecutive years (2017-2018 and 2018-2019), thus making four environments . The panel was genotyped for 12,196 SNPs based on DArT-seq (outsourced to DArT Ltd by CIMMYT); these SNPs included 5,400 SNPs, which could not be assigned to individual chromosomes and were therefore, described as unassigned by the vendor. Phenotypic data was recorded on the following three disease-related traits: (i) Area Under Disease Progress Curve (AUDPC), (ii) Incubation Period (IP), and (iii) Lesion Number (LN). GWAS was conducted using each of five different models, which included two single-locus models (CMLM and SUPER) and three multi-locus models . This exercise gave 306 MTAs, but only 89 MTAs  including a solitary MTA detected using all the five models and 88 identified using four of the five models (barring SUPER) were considered to be important. These were used for further analysis, which included identification of candidate genes (CGs) and their annotation. A majority of these MTAs were novel. Only 70 of the 89 MTAs were assigned to individual chromosomes; the remaining 19 MTAs belonged to unassigned SNPs, for which chromosomes were not known. Seven MTAs were selected on the basis of minimum P value, number of models, number of environments and location on chromosomes with respect to QTLs reported earlier. These 7 MTAs, which included five main effect MTAs and two for epistatic interactions, were considered to be important for marker-assisted selection (MAS). The present study thus improved our understanding of the genetics of resistance against spot blotch in wheat and provided seven MTAs, which may be used for MAS after due validation. Wheat is the third most important crop world-wide, next only to rice and maize, contributing ~20% of total dietary calories and proteins worldwide . AlthougIt is widely known that the use of resistant cultivars is the safest means to safeguard against yield losses and is also environmentally safe. A knowledge of the genetics of resistance in the host is necessary for the development of these resistant cultivars. Therefore, a large number of studies for the study of the genetics of resistance have been undertaken in the past. The early inheritance studies suggested monogenic to polygenic resistance with the involvement of dominant as well as recessive genes. The availability of dominant as well as recessive nature of disease resistance in these different studies has been attributed to the use of parents with different genetic constitutions and the possibility of same genes behaving as dominant in one genetic background and recessive in the other . The four Sb genes in the former category included the following: Sb1 (7DS), Sb2 (5BL), Sb3 (3BS) and Sb4 (4BL)  . As a qub4 (4BL) , but no is known .A number of interval mapping studies for the identification of QTLs and GWA studies for identification of MTAs have also been conducted  has also been studied for occurrence of races/pathotypes and identification of genes involved in pathogenesis. In several studies, isolates have been collected from specific geographical regions and differentials suggested for classification of these isolates into groups, sometimes erroneously described as pathotypes  has been identified. However, a virulence gene ToxA in the pathogen and its sensitivity gene Tsn1 in the host following IGFG for spot blotch was identified recently  and Tsn1 gene in 36.8% of wheat genotypes (81 of 220 wheat genotypes). Whole genome sequence of the pathogen (B. sorokiniana) has also been worked out recently and putative avirulence genes suggested, but these genes need to be validated through further investigations  and to detect loci which provide spot blotch resistance through a novel association panel (never used earlier). As expected, many novel MTAs involved in spot blotch resistance were identified in the present study. The results of the study are presented and discussed in this communication.The GWAS panel consisted of 303 diverse wheat accessions  unique mapping to an unambiguous locus; (ii) maximum 1 bp mismatch to the marker sequence, and (iii) markers with multiple alignment options discarded, if the second-best hit showed < 3 bp mismatch to the marker sequence, i.e. markers with 2 or more hits (loci) were discarded if there was not at least 3 bp difference between the best and second-best hit. (iv) Monomorphic markers were discarded as well as (v) SNPs with MAF (Minor Allele Frequency less than 5% and more than 5% missing data).The panel was raised in a CRBD with two replications in each of the following four environments: E1 (2017-2018) and E2 (2018-2019) at Agriculture Research Farm, BHU, Varanasi, UP, E3 (2017-2018) and E4 (2018-2019) at BISA Agriculture farm, Pusa, Samastipur, Bihar. Recommended crop management practices were followed . Each genotype in a replication was represented by a plot of 3 rows of 1m each, with a row-to-row distance of 0.25 m.Pure culture of a highly aggressing isolate of the pathogen  was multiplied on sorghum grain and used for inoculation following For AUDPC, disease severity (%) was recorded in three different growth stages (GS), GS 63 , GS 69 (anthesis complete) and GS 77 (late milking). Disease severity was assessed by the formula [(1/9 (D1×D2) ×100] using the double-digit scale  . The firi and Y (i + 1) = disease severity at time ti and t (i + 1), respectively; t (i + 1)—t i = time interval (number of days) between two disease scores assessed.Where YIP was recorded as the duration (in number of days) from inoculation to the appearance of visible symptoms on five randomly tagged plants in each plot . SimilarANOVA and correlation coefficients were obtained using Agricolae package in R studio. Violin plots for phenotypic data were developed for each of the four individual environments and BLUP values. For this purpose, BLUP values were obtained using the ‘lme4’ in R . DescripGenotype data were available for 17,937 SNPs, but only 12,160 markers remained after filtering out those with a marker allele frequency (MAF) of less than 5%. These 12,160 SNPs were then employed in PCA/population structure analysis and GWAS.The principal component (PC) analysis using genotypic data was conducted for the development of population structure (Q matrix) and relatedness (K matrix) using tools available in GAPIT ; Q and KPopulation structure involving 210 SNPs (ten SNPs from each chromosome) was examined using the software STRUCTURE version 2.3.4 . The detFollowing five different models were used for GWAS: (i) Compressed mixed linear model CMLM; ; (ii) Se2  was carried out by using PLINK2 . It is f2 (-8 . The SNP3. Highly significant annotated CGs were retrieved from a 200 kb window for each MTA. IWGSC4 was used for gene ontology (GO) annotation information of these CGs.The most significant MTAs detected were also used for identification of CGs through alignment of sequences associated with MTAs with wheat genome assembly IWGSC v.1  are presented in A representative set of Manhattan plots and QQ plots based on BLUP data are depicted in MTAs representing 44 pairs of first-order epistatic interactions (SNP × SNP interactions) are listed in When compared with 84 known QTLs reported in earlier studies, only seven MTAs of the 70 (remaining 19 were unassigned) occurred within the QTL interval (in green colour)  and the other 12 occurred in the vicinity of the markers flanking  the QTLs reported earlier Figure 5Genomic regions within a window of 200 kb of each MTA (100 kb on each side), when subjected to identification of CGs, gave 163 CGs . These CGs were associated with only 72 MTAs ; the remaining 21 MTAs gave no CGs. These CGs, when screened for the identification of genes already known to be involved in different pathways of pathogen–host interactions and pathogenesis, gave 64 CGs, which included 25 CGs for AUDPC, 23 for IP and 16 for LN  was evaluated for variation in spot blotch resistance at two different locations of India, which represented regions with warm humid climate, suitable for the spot blotch disease. The study allowed identification of genomic regions carrying markers associated with functional loci for SB resistance. High level of variability  for each of the three traits suggested that the panel was suitable for a study of the genetics of quantitative traits. The same panel was earlier utilized by us in GWAS for several other traits including the following: (i) yield related traits ; (ii) FeThe genotyping data for 210 SNP markers (distributed on 21 chromosomes) suggested a low level of population structure in the association panel Figure 2In the present study, 306 MTAs were identified using one or more of the five models used in the present study. There was a solitary MTA , which was detected by all the five models. The remaining 305 MTAs were first placed in two groups, those identified using SUPER and those common to all the remaining four models. The MTAs other than 88 MTAs common to four models, were then classified into MTAs common to three models, two models and those unique to individual models  in the last three decades. A variety of molecular markers and statistical tools were utilized for the earlier studies involving all major crops. At least >30,000 QTLs for different traits including yield and tolerance to biotic and abiotic stresses are already available in wheat  has several limitations . These tEpistatic interactions are often ignored in GWAS, although recently studied for the following traits: (1) flowering time , (2) steAmong the MTAs identified in this study, 13 MTAs suitable for marker-assisted selection (MAS) were initially selected using the following criteria: (1) lowest P-value, (2) identified by more than one models, (3) identified in more than one environment, (4) reported in earlier studies. These 13 MTAs included five MTAs for AUDPC, two MTAs for IP, six MTAs for LN Table 4.Puccinia triticina causing leaf rust (Zymoseptoria tritici causing septoria tritici blotch (STB) and some other fungal diseases  sediseases ; (iii) pdiseases ; (iv) sadiseases .


HPV detection has been proposed as part of the co-testing which improves the sensitivity of cervical screening. However, the commercially liquid-based medium adds cost in low-resource areas. This study aimed to evaluate the performance of ice-cold phosphate buffer saline (PBS) for HPV detection. HPV DNA from SiHa cells (with 1-2 copies of HPV16 per cell) preserved in ice-cold PBS or PreserveCyt solution at different time points  was tested in triplicate using Cobas 4800. The threshold cycle (Ct) values of both solutions were compared. An estimated false negative rate of PBS was also assessed by using the difference in Ct values between both solutions (∆Ct) and Ct values of HPV16-positive PreserveCyt clinical samples (Ctsample) at corresponding time points. Samples with a (Ctsample+∆Ct) value > 40.5 (the cutoff of HPV16 DNA by Cobas 4800) were considered as false negativity. The Ct values of HPV16 DNA of SiHa cells collected in PBS were higher than PreserveCyt ranging from 0.43 to 2.36 cycles depending on incubation times. There was no significant difference at 24, 72, 120, and 168 h. However, the Ct values were statistically significantly higher for PBS than PreserveCyt at 36 h (31.00 vs 29.26), and 48 h (31.06 vs 28.70). A retrospective analysis in 47 clinical PreserveCyt collected samples that were positive for HPV16 DNA found that 1 case (2%) would become negative if collected in ice-cold PBS. The PBS might be an alternative collecting medium for HPV detection in the low-resource areas. Further evaluations are warranted. Cervical cancer is the fourth most common cancer among women worldwide . In Thailand, cervical cancer is the second most common cancer in women in Thailand . In 2012, 8,184 new cases of cervical cancer and 4,513 deaths in Thai women were estimated by the ministry of public health and ministry of education . According to data from the National Cancer Registry, the age-standardized incidence rate (ASR) for cervical cancer in Thailand was 11.7-14.4 per 100,000 . Cervical cancer is highly related to human papillomavirus (HPV), especially high-risk HPV (hrHPV) group . Molecular testing has been developed for detecting the HPV viral nucleic acid in clinical samples. The HPV DNA detection with a high sensitivity has improved the performance of cervical cancer screening . Therefore, the HPV DNA detection has been recommended to use with liquid-based cytology as a co-test in the primary cervical cancer screening program .The Cobas 4800 HPV DNA test was approved by the United States Food and Drug Administration in 2011 for detecting hrHPV DNA. This test is based on real-time polymerase chain reaction (PCR) technology and nucleic acid hybridization that detects 14 hrHPV types: HPV type 16, 18 and a pool of 12 other hrHPV types  . Cervical cells were recommended to be collected in ThinPrep PreserveCyt solution, Cobas PCR cell collection media and SurePath preservative fluid. PreserveCyt, a widely used preservative for liquid-based cytology in cervical cancer screening , is a methanol based buffer which can preserve cell morphology as well as DNA for molecular study . However, this preservative solution adds a substantial cost for low-resource areas that need hrHPV nucleic acid testing.Phosphate buffer saline (PBS) is an isotonic solution commonly used in laboratory especially for cell culture application. PBS is nontoxic to living cells and can retain cell viability during transportation . Due to its economical cost and simple preparation, we hypothesized that PBS may be an alternative collecting medium for HPV DNA detection under temperature and time control. Therefore, this study aimed to evaluate whether ice-cold PBS could be used as a collecting medium for HPV DNA detection in SiHa cells at different collected time points, compared with PreserveCyt solution. In case that the performance of ice-cold PBS is inferior to PreserveCyt, an estimated false negative rate of PBS was also assessed by using data from clinical samples that were collected in PreserveCyt and were positive for HPV16 DNA by Cobas 4800 HPV DNA test.Cell culture2 incubator until reaching 90% confluence. The SiHa human cervical cancer cell line containing 1-2 copies of HPV DNA type 16 per cell was cultured in DMEM  supplemented with 10% FBS  in T-25 flask . Cells were incubated at 37°C in 5% COSiHa cell preparation for HPV DNA detection 5 SiHa cells were placed in 2 ml of ice-cold PBS or PreserveCyt solution  for various incubation times . For each time point, both PBS and PreserveCyt samples were prepared in triplicate. Then, HPV DNA detection was performed simultaneously using Cobas 4800 HPV DNA test according to manufacturer recommendations. After harvesting, 2x10Briefly, samples were processed for DNA extraction using the Cobas X480 instrument then DNA was purified by magnetic beads system. The purified DNA was transferred into the Cobas Z480 for PCR amplification with subsequent nucleic acid hybridization. The human β-globin gene was used as an internal control for the quality of DNA extraction. Test was validated with positive and negative control in each run. Results were interpreted as negative or positive for HPV16, HPV18 or other hrHPVs. Test with no β-globin gene amplification and negative HPV DNA was interpreted as invalid.Retrospective analysis for estimating the performance of PBS in clinical samplesThe retrospective study population was part of 5,331 women enrolled in the population-based cervical cancer screening program in Northern Thailand during January 2014 to December 2015. All samples in this program were collected in PreserveCyt and tested for hrHPV DNA by Cobas 4800 HPV DNA test. There were 79 women who were positive for HPV16 DNA. Among these, 47 had time from collection to test at 1, 2, 3, 5 and 7 days. An estimated Ct value of HPV16 DNA if using ice-cold PBS was calculated in each case as follow;Estimated Ct value = Ctsample + ∆Ct (corresponding time point)where,Ctsample = Ct values of HPV16-positive PreserveCyt clinical samples∆Ct = difference in Ct values between PBS and PreserveCyt solution from the SiHa experiment at corresponding time pointEthical considerationsThis study was approved by the institutional ethic committee of the Faculty of medicine, Chiang Mai University in study code: FAC-MED-2561-05275.Statistical analysisAbsolute Ct values from each triplicate experiment were collected. Unpaired t-test was used for comparisons between solutions at each time points using GraphPad Prism software version 7. A P value of < 0.05 was considered statistically significant. Ct values of SiHa cells at different incubation timesThe Ct values of HPV16 DNA ranged from 30.13 to 32 for ice-cold PBS and 28.7 to 31.06 for PreserveCyt . They weEstimated false negative rate of ice-cold PBS in clinical samples To estimate a false negative rate of ice-cold PBS in clinical samples, 79 women with HPV16-positive PreserveCyt collected samples were retrieved from a total of 5,331 tested samples during January 2014 to December 2015. The Ct values of these 79 samples ranged from 20.1 to 40.5 for HPV16 DNA and 25.7 to 39.2 for β-globin gene . Forty-seven of 79 samples had collection-to-test time points corresponding to the SiHa experiment which were 1, 2, 3, 5 and 7 days. Estimated Ct values in these samples if using ice-cold PBS were calculated by summing a ∆Ct at the corresponding time point and Ctsample . Since the cutoff value of HPV16 by Cobas 4800 HPV DNA test is set at 40.5 , any samples with an estimated Ct value higher than 40.5 were considered as a false negative sample. There was only 1 out of 47 samples (2%) expected to be negative for HPV16 in case of using PBS . The HPV DNA detection has been used in combination with cervical cytology for primary cervical cancer screening . Liquid-based cytology samples are recommended for most approved HPV DNA detection kits. However, adding cost of commercial collecting media for liquid-based cytology can be substantially problematic in low-resource areas. Low-cost liquid medium for HPV DNA detection has been evaluated by Castle et al., (2007). They suggested that the mouthwash was as alternative low-cost liquid medium for storage of cervical specimens for HPV DNA detection . However, the mouthwashes are available in various and different in the composition depending on the company. Since PBS is commonly used in laboratory, cheap, easy to prepare and non-toxic to cells, the present study therefore hypothesized that ice-cold PBS might be used as an alternative cell collecting medium for HPV DNA detection by Cobas 4800 HPV DNA test. The present study demonstrated that HPV DNA detection of SiHa cells collected in ice-cold PBS was comparable to the reference liquid-based medium PreserveCyt. For the SiHa experiment, there was no significant difference in Ct value of the internal control β-globin gene between ice-cold PBS and PreserveCyt at every time point. This confirms that ice-cold PBS can preserve the SiHa cells similar to PreserveCyt up to 7 days. The performance of ice-cold PBS appeared to be inferior to PreserveCyt with regard to HPV DNA detection at 36 h and 48 h however there was no significant difference during 72-168 h. Given that an increase in Ct value of HPV16 DNA if using ice-cold PBS instead of PreserveCyt ranged from 0.43-2.36 cycles, any sample with HPV DNA copy number of 20.43-2.36 or 1.35-5.13 times of the limit of detection may become negative if using PBS as a collection media. However, the present study also demonstrated the estimated false negative rate of only 2% from a retrospective analysis of clinical samples in the past 2 years. Banura et al., (2008) evaluated the performance of PBS in the detection of 25 HPV types compared with paper smear using SPF10 HPV LiPA version 1 . The prevalence of any type HPV of the cervical exfoliated cells collected in PBS showed much higher than paper smear  . This study demonstrated that ice-cold PBS could be used to preserve DNA in tissue culture cells for HPV detection. The finding suggests that the PBS is has a potential to be used as a collection media for cervical cancer screening samples, particularly in low-resource settings. As this study was based only on culture cell samples, the comparison of clinical performance in HPV DNA testing between PBS and standard preservative solution was not available. Further study in clinical cervical samples is needed in the comparative evaluation of the performance of PBS including sensitivity, specificity, false positive, and false negative values.In summary, our study provided preliminary evidence that HPV DNA detection can be performed by using ice-cold PBS. The ice-cold PBS would be beneficial for HPV DNA detection in low-resource areas still using conventional Pap smear because its cost per test was approximately 8 times lower than PreserveCyt . Limitation in the use of PBS should be addressed as PBS needs to be preserved in cold temperature and the samples needs to be processed within the time limit (one week after collection). Thus, clinical application of PBS would require a well-organized arrangement. However, PBS is cost-saving compared to the standard collection media for HPV testing, and this can be beneficial to cervical cancer screening in low-income areas.WD: conceptualized and designed the study, conducted data collection, analyzed and interpreted the data, wrote the first draft of the manuscript, and approved the final version to be published; SK: critically reviewed the manuscript; SS: critically reviewed the manuscript; AS: conducted data collection; PA: conducted data collection; SL: conceptualized the study, analyzed and interpreted the data, and critically reviewed the manuscript; All authors reviewed the results and approved the final version of the manuscript.This work was supported by the National Research University Project which was authorized by the Higher Education Commission of Thailand and Faculty of Medicine, Chiang Mai University.


Atrophy of the reproductive tract mucosa caused by the decrease of estrogen may increase the detection rate of ASC-US in cervical cytology of post-menopausal women. In addition, other pathogenic infections and inflammation can change the cellular morphology and increase the detection rate of ASC-US. However, further studies are needed to elucidate whether the high detection rate of ASC-US in post-menopausal women leads to the high referral rate of colposcopy.This retrospective study was conducted to document ASC-US in cervical cytology reports at the Department of Cytology at Gynecology and Obstetrics, Tianjin Medical University General Hospital between January 2006 and February 2021. We then analyzed 2,462 reports of women with ASC-US at the Cervical Lesions Department. A total of 499 patients with ASC-US and 151 cytology with NILM participants underwent vaginal microecology tests.The average reporting rate of ASC-US in cytology was 5.7%. The detection rate of ASC-US in women aged > 50 years (7.0%) was significantly higher than that in women aged ≤50 years (5.0%) (P<0.05). The CIN2+ detection rate was significantly lower in the post- (12.6%) than in pre-menopausal (20.5%) patients with ASC-US (P <0.05). The prevalence of abnormal reporting rate of vaginal microecology was significantly lower in the pre-menopausal group (56.2%) than that in the post-menopausal group (82.9%) (P<0.05). The prevalence of bacterial vaginosis (BV) (19.60%) was relatively high in the pre-menopausal group, but the abundance of bacteria-inhibiting flora (40.79%) was mainly an abnormality in the post-menopausal group. The vaginal microecological abnormality rate of the women with HR-HPV (-) of ASC-US was 66.22%, which was significantly higher than that of the HR-HPV (-) and the NILM group .The detection rate of ASC-US in women aged > 50 years was higher than that ≤50 years, but the detection rate of CIN2+ was lower in the post-menopausal women with ASC-US. However, vaginal microecological abnormalities may increase the false-positive diagnosis rate of ASC-US. The vaginal microecological abnormalities of the menopausal women with ASC-US are mainly attributed to infectious diseases such as BV, and it mainly occurs in the post-menopausal women was bacteria-inhibiting flora. Therefore, to avoid the high referral rate for colposcopy, more attention should be paid to the detection of vaginal microecology. The National Cancer Center (NCC) of China reported in the 2016 nationwide statistics for cancer that the incidence and mortality rates of cervical cancer were ranked the fifth and seventh among all the female malignancies, respectively, and these rates continue to be on the rise . TherefoLactobacillus , vulvovaginal candidiasis (VVC), trichomoniasis and aerobic vaginitis (AV) are associated with an increased risk of CIN . Liu et bacillus . It has bacillus .In the current study, we document and analyze the reporting rates of the age-stratified ASC-US, the immediate histopathology of ASC-US along with HR-HPV genotype distribution, outcomes of post-menopausal women with ASC-US, and the correlation between vaginal microecology and ASC-US from the department of Gynecology and Obstetrics, Tianjin Medical University General Hospital. This aims at offering new parameters to design an optimal strategy for the triage of pre- and post-menopausal patients with ASC-US.22.1This retrospective study was conducted to document the cervical cytology reports of patients with ASC-US at the Department of Cytology at Gynecology and Obstetrics, Tianjin Medical University General Hospital (TMUGH) between January 2006 and February 2021. We then analyzed 2,462 reports of women with ASC-US who underwent HPV genotyping, colposcopy, and histopathological examination at the Cervical Lesions Department of Obstetrics and Gynecology between January 2010 and December 2021. It is important to emphasize that a total of 499 participants with ASC-US and 151 with HR-HPV-negative and NILM cytology underwent vaginal microecology tests. The inclusion and exclusion criteria are shown in 2.2The Liquid-based cytological test (LCT) method was used in this study. LCT preparation was performed by Becton, Dickinson and Company. Cytological evaluation was performed by two cytopathologists at the Department of Cytology at Gynecology and Obstetrics, TMUGH. Cytological results were classified using the 2001 TBS criteria for reporting cervical cytology before 2014 , and the2.3The HR-HPV genotypes  were detected using a polymerase chain reaction-reverse dot blot (PCR-RDB) HPV genotyping kit . The procedures were performed in accordance with the manufacturer’s instructions. A small number of patients were tested for HPV in other hospitals, and the HPV test was performed using the Roche Cobas 4800 HPV DNA and HC2 tests. The Roche Cobas 4800 HPV DNA test can detect 14 HR-HPV types, including HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68. HC2 test can be used for the quantitative detection of 13 high-risk HPV types, without HPV genotype, and ≥1.0pg/mL is considered positive.2.4ASC-US with positive HR-HPV or negative HR-HPV and more than twice ASC-US cytology were referred for colposcopy for biopsy. Colposcopy and biopsy were performed by experienced gynecological specialists from TMUGH. The duration between cytological sampling and colposcopy did not exceed 60 days. During the colposcopy, all the visually abnormal areas were biopsied. Quadrants with normal colposcopic impressions were biopsied at the squamocolumnar junction (“random biopsy”). Endocervical curettage(ECC)is performed when the TZ is not visible or fully visible. All histological slides were reviewed by two gynecological pathologists who were blinded to the cytology results at TMUGH. Immunohistochemistry was used to adjudicate difficult or equivocal diagnoses.2.5Lactobacillus morphotypes (scored as 0–4), G. vaginalis morphotypes (scored as 0–4), and Mobiluncus morphotypes (scored as 0–2). A Nugent score of 7–10 was interpreted as consistent with BV, a score of 4–6 as intermediate (BV intermediate type), and a score of 0–3 as negative for BV. Wet-mount microscopy has traditionally been used as the preferred diagnostic test for trichomoniasis in women. A diagnosis of Candida vaginitis is indicated by a wet preparation  of vaginal discharge demonstrating budding yeasts, hyphae, or pseudohyphae  and microscopic examinations. Vaginal discharge smears were spread on 3 glass slides for immediate microscopic examination. AV, BV, Trichomoniasis, and VVC were defined according to their recognized standards. The Nugent score was used to diagnose BV and was calculated by assessing the number of dohyphae . The diadohyphae  by microdohyphae . The res2.6P < 0.05 was considered statistically significant. Cochran and Mantel-Haenszel tests were performed to evaluate the common odds ratio (OR) of the HPV-positive group compared with the HPV-negative group, controlled by pre- and post-menopause. A P-value < 0.05 was considered statistically significant.The statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 25.0 software for windows . Chi-square test or Fisher’s exact test was used for analyzing categorical variables. Statistical tests were two-sided, and 33.1P <0.05). The ASC-US reporting rates of 7.3%, 7.1%, and 7.1% of in the age groups of 51–60 years, 61–70 years, and below 20 years, respectively, were significantly higher than the reporting rates of 5.4%, 5.1%, 4.9%, 4.5%, and 4.4% observed in the age groups of 41–50 years,71–80 years, 31–40 years, 21–30 years, and > 80 years, respectively (P < 0.05).During the 16-year retrospective study period, a total of 270,682 gynecologic LCT tests were performed (148 patients have been excluded due to the lack of their age-related information). ASC-US cytology was the most frequent abnormal interpretation, accounting for 5.7% of all cervical cytology in the survey Table 1.3.2A total of 2,462 eligible patients had confirmed ASC-US by cytology and were included in the study between January 2010 and December 2021, with an average age of 43.06 ± 12.27 years . The overall HPV test results revealed that 2,381 patients had the HPV genotype, 39 had Cobas 4800, and 42 had HC2. Ultimately, there were 1,965 patients with HPV-positive status and 497 patients with HPV-negative status. The histological analysis confirmed that 928 patients (37.7%) had normal cervixes, 1,085 (44.1%) had CIN1, 145 (5.9%) had CIN2, 207 (8.4%) had CIN2–3, 78 (3.2%) had CIN3, 3(0.1%) had AIS, and 16 (0.6%) had carcinoma (including 15 squamous cell carcinomas and 1 adenocarcinoma). Among the 2,462 patients with histopathological results, diagnoses of CIN2+ were reported in 449 patients (18.2%).2 = 21.224, P=0.000, <0.05).The CIN2+ detection rate was significantly lower in the post- than in the pre-menopausal patients with ASC-US, as demonstrated in 2 = 68.470, P=0.000, <0.05).  of the women with ASC-US/HPV-positive results which is significantly higher than the rate of 5.4% for women (27 of 497) with ASC-US/HPV-negative results (X <0.05). Table 3.P <0.05). Furthermore, in the patients with ASC-US/HR-HPV-negative status, there was no significant difference in the detection rate of CIN2+between the post- and pre-menopausal women . The detection rates of CIN2+ in the HR-HPV-positive group were significantly higher than those in the HPV-negative group in both the pre-menopausal or post-menopausal women with ASC-US .In the patients with ASC-US/HR-HPV-positive status, the CIN2+ detection rate was significantly lower in the post- than in pre-menopausal patients with ASC-US, as demonstrated in P <0.05). Furthermore, the CIN2+ detection rate of the HPV16/18 positive patients with ASC-US was significantly higher than that of the other HR HPV-positive groups, P < 0.05. The detailed results for the pre-and post-menopausal women with ASC-US are provided in P < 0.05. However, the detection rates of CIN2+ in the pre- and post-menopausal HPV18 positive group were similar (P >0.05).A total of 475 (19.6%) patients were HPV16 positive, 147 (6.1%) were HPV18 positive, 25 (1.0%) were HPV16 and HPV18 positive, and 1,276 (52.7%) were HR HPV-positive. HPV16, HPV16 and 18 positive both showed the severity of the histological diagnosis, and the rate of CIN2+ was significantly higher than the HPV18 positive group and the HR HPV-positive group (P > 0.05), but significantly higher than that of HPV52, 58, and 18 (P < 0.05). The detection rate of the CIN2 + lesions from the five most common HR-HPV genotypes  in women was 25.6% (227/885), which was significantly higher than that of the other single HR-HPV types .In the patients with ASC-US with a single infection, HPV16 was the most common HR-HPV genotype, followed, in order of decreasing frequency, by HPV52, 58, 18, and 33. The detection rate of CIN2+ in HPV16 (40.9%) was similar to that of HPV33 . The detection rate of CIN2+ in HPV52 pre-menopausal patients was significantly higher than that in the post-menopausal patients (P < 0.05)  Table 6.3.3P<0.05; P>0.05).In total, 499 patients underwent vaginal microecology analysis among the 2,462 patients with ASC-US in this study. In general, the abnormal reporting rate for vaginal microecology was 64.3% (321/499). The prevalence of abnormal reporting was significantly lower in the pre-menopausal group (56.2%) than that in the post-menopausal group . However, the prevalence of VVC in the pre-menopausal group was significantly higher than that in the post-menopausal group (P<0.05). The prevalence of AV, bacteria-inhibiting flora, and atrophic vaginitis in the post-menopausal patients was significantly higher than that the in pre-menopausal patients (P < 0.05).In the present study, the abnormal vaginal microecology rate of BV was the highest, accounting for 18.6% (188/448). However, the distribution of vaginal microecological abnormalities was different in the pre- and post-menopausal groups, and the prevalence of BV (19.6%), BV intermediate type (9.8%), and mixed vaginitis were relatively high in the premenopausal group, but abnormalities in the bacteria-inhibiting flora were mainly evident in the post-menopausal group, accounting for 40.8% Table 8.P <0.048), and the false-positive rate of ASC-US diagnosis increased due to the abnormal microecology. However, there was no significant difference in the rate of microecological abnormalities between the post-menopausal and pre-menopausal women.To further explore whether the vaginal microecological abnormalities can lead to a false positive diagnosis of ASC-US, we collected the vaginal microecological results of healthy women with negative HPV and NILM in the same period, including 92 pre-menopausal and 59 post-menopausal womenTable 9.4ASC-US is the most common finding among the epithelial cell abnormality, but has an equivocal cytologic diagnosis. The histopathology results of ASC-US are very different and can either be an actively proliferated benign lesion or a potentially malignant lesion .ASC-US can be characterized by (1) squamous differentiation, (2) increased nuclear-to-cytoplasmic ratio, and (3) minimal nuclear changes that may include hyperchromasia, chromatin clumping, irregularity, smudging, and/or multinucleation. Subtle and subjective findings in the specimens with ASC-US have resulted in poor reproducibility, thus compounding the difficulty in developing and illustrating strict criteria. In the case of the presence of reactive/reparative or degenerative changes, organisms, air-drying with artifactual nuclear enlargement, atrophic patterns, and atrophy of other artifacts, it is difficult to diagnose NILM or ASC-US. Thus, referral to the patient’s age, medical history, and HPV results may be needed. In this study, the detection rate of ASCUS and the results of HPV, histopathology, and vaginal microecology were analyzed.In this study, the detection rate of ASC-US was 5.7%, which is within the range of 3.7–10% of the ASC-US reporting rate in the Chinese population . With agIn this study, the histopathological correlation indicated that CIN2+ and CIN3 were identified in 18.2% of the women who had ASC-US cytology. In a pooled analysis, the CIN2+ detection rate of ASC-US was 3.2% in the women aged 15–59 for initial screening, but the risk ratio greatly increased with the increase in the cumulative time and in the participants in the clinics or hospitals . A studyIn the recent years, HR-HPV testing has been included in cervical cancer screening programs, which can be used to triage patients with ASC-US. According to the most current ASCCP risk-based management consensus guidelines, the immediate risk of CIN3+ is 4.4% for ASC-US/HPV-positive and 0.04% for ASC-US/HPV-negative populations . Zheng eAccording to the principle of “equal management of equal risk”, patients with ASC-US with different HPV genotypes should be treated differently in the clinical management. A systematic review conducted by Bonde et al. demonstrated that published evidence supports the clinical utility of HPV genotyping in risk discrimination for CIN3+ lesions during cervical cancer screening in the US, United Kingdom, Sweden, Denmark, and the Netherlands . RecentlLactobacillus. The increased diversity of the bacterial community may cause upper genital tract pathological infections. Cervicitis and vaginitis have been reported as major confounding factors for the diagnosis of squamous epithelial cell abnormality (The vaginal microecology is a significant niche of the human microbiome, and the vaginal microbiota plays a significant role in the wellness maintenance of the female reproductive tract. When the vagina cannot maintain its microecological balance, the acidic environment of the vagina is destroyed, and the proliferation of pathogenic bacteria replaces the initial dominance of ormality . To explormality . Paba etormality . Our stuLactobacillus and facilitates the survival of pathogenic bacteria and viruses (Overall, in our study, BV was the most common microecological abnormality, which is in agreement with the previous literature . However viruses . In addiThe strength of this study is that it is the first large-scale retrospective analysis to demonstrate differences in the distribution of high-risk HPV genotypes and histopathology for ASC-US in pre- and post-menopausal women. We also demonstrated an association between the vaginal microecology and ASC-US diagnosis. However, the current study had some limitations. First, this was a retrospective study, and not all women with ASC-US were referred for colposcopic biopsy. In our study, HPV positive/ASC-US represented the majority of the patients. Accordingly, the detection rate of CIN2+ was high, which could have introduced bias in the results. In addition, not all the women in the ASC-US group had vaginal microecology, and we did not perform a stratified analysis of the symptoms of the patients of vaginitis. In future studies, the influence of HPV should be excluded and the influence of vaginal microecology on the women with ASC-US/HPV should be analyzed. Second, we did not consider the impact of HPV vaccines in this study population. However, bivalent vaccines were only approved in China in 2016; therefore, the impact of vaccines in this study population is small, but it should be a focus for further stratified analysis in the future. Therefore, large-scale prospective studies are needed to verify the distribution of HPV genotypes in women with ASC-US, differences in ASC-US among the pre- and post-menopausal women, and the impact of vaginal microecology on the diagnosis of ASC-US.In conclusion, the detection rate of ASC-US in women over 50 years old was higher than that in women less than 50 years old, but the detection rate of CIN2+ was lower in the post-menopausal women than in the premenopausal women with ASC-US. However, the vaginal microecological abnormalities may increase the false-positive diagnosis rate of ASC-US. The vaginal microecological abnormalities of menopausal women with ASC-US are mainly infectious diseases such as BV which occurs in post-menopausal women and are mainly caused by the decrease in the bacteria-inhibiting flora due to mucosal epithelial atrophy. Therefore, more attention should be paid to the detection of vaginal microecology to avoid the high referral rate for colposcopy.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 Ethical Committee Tianjin Medical University General Hospital Tianjin, China. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AF, and FX conceived the study question, and all authors were involved in the study design. YY was involved in the statistical analysis while BL, LD, and CW interpreted the results. BL created the first draft of the manuscript. BL, LD, CW, JL, XZ, AF, and FX made substantial contributions to drafting the article and revising it critically. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Primary prophylactic, early detection and the treatment of precancerous lesions are the main goals of cervical cancer screening. Despite effective surgical treatment methods, using loop electrosurgical excision procedures and conization, the overall risk of the recurrence of HSIL lesions remains at approximately 6.6%. There is increasing evidence of the potential role of HPV vaccines in the adjuvant setting and their impact on the reduction of disease recurrence. This review aims to analyze the up-to-date research concerning the use and efficacy of secondary human papilloma virus (HPV) vaccination as an adjuvant method to surgical treatment in patients diagnosed with cervical HSILs.Cervical cancer formation is preceded by precursor lesions, including low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs), which are usually diagnosed in women of reproductive age. Despite the recent advanced diagnostic and treatment methods, including colposcopy, the loop electrosurgical excision procedure (LEEP), and surgical conization, the recurrence or residual disease affects as many as 6.6% of patients. The lesions are often associated with human papilloma virus (HPV) infection. As HPV persistence is the leading and only modifiable factor affecting the risk of progression of CIN lesions into high-grade cervical dysplasia and cancer, it has been proposed to conduct adjuvant vaccination in patients treated for high-grade cervical dysplasia. To date, no vaccine has been approved for therapeutic use in patients diagnosed with HSILs; however, attempts have been made to determine the use of HPV prophylactic vaccination to reduce recurrent HSILs and prevent cervical cancer. The aim of this review was to analyze the up-to-date literature concerning the possible use of secondary human papilloma virus (HPV) vaccination as an adjuvant method to surgical treatment in patients diagnosed with cervical HSILs. Adjuvant HPV vaccination after surgical treatment may reduce the risk of recurrent cervical dysplasia. Each year, more than half a million women are diagnosed with cervical cancer . DespiteCervical cancer is preceded by precursor lesions, including low-grade squamous intraepithelial lesions (LSILs) and high-grade squamous intraepithelial lesions (HSILs), which are usually diagnosed in women of reproductive age. Despite the recent advanced diagnostic and treatment methods, including colposcopy, the loop electrosurgical excision procedure (LEEP), and surgical conization, the recurrence or residual disease affects as many as 6.6% of patients .HPV is a family of more than 200 types of non-enveloped double-stranded DNA viruses that can infect mucosal and cutaneous epithelial cells. They can be divided into high risk and low risk based on their oncogenic capacity. The most common high-risk viruses responsible for approximately 70% of persistent HPV infections are HPV types 16 and 18, while types 31, 33, 45, 52, and 58 account for 19% ,11. HoweThe HPV genome contains early (E) genes, which control DNA maintenance, replication, and transcription, while the late (L) genes encode proteins that build up the viral capsid. In the early stages of HPV infection, proteins E1 and E2 are expressed in significant amounts and allow viral replication within the infected tissues. In cervical HPV infection, the changes lead to the induction of low-grade squamous intraepithelial lesions. The best known mechanism responsible for malignant transformation involves viral oncoproteins E6 and E7, as they bind to the p53 tumor suppressor protein and retinoblastoma tumor suppressor protein (pRb), leading to the degradation of the suppressor proteins and allowing for cell proliferation and the initiation of carcinogenesis . The intHPV infection occurs via a microinjury from an infection or sexual contact when antigen-presenting cells (APCs) are exposed to the HPV virus. Monocyte–macrophage and dendritic cells localized in the epithelium induce immune responses by releasing proinflammatory cytokines, including interleukins IL-1, IL-6, and IL-12, and tumor necrosis factor-alpha (TNF-α) . Host inThe primary prevention of cervical cancer is achieved by avoiding HPV infection. The most effective method relies on HPV vaccination, especially in adolescents before their first sexual encounter. Currently, there are three vaccines approved by the U.S. Food and Drug Administration (FDA) that can be administered: bivalent (Cervarix), quadrivalent (Gardasil), and nonavalent (Gardasil 9) HPV vaccines. The first vaccines became available in 2006 and demonstrated a 90% efficiency rate in HPV infection prevention with HPV virus types 16 and 18 . AdditioAll vaccines were developed using recombinant DNA technology. The vaccines were created from the purified self-assembling L1 protein, which forms HPV type-specific empty shells to mimic virus-like particles. As a result, the vaccines have high immunogenic potential and induce the organism production of HPV-specific antibodies, effectively preventing viral infection . The vacThe vaccines were found to reduce the risk of HPV infection, the development of HPV-related lesions, and the rates of relapse and subsequent HPV-related diseases after surgery for HPV-related diseases of the cervix and vulva. A post hoc analysis conducted by Joura et al. , based oThe early detection and treatment of precancerous lesions are the methods of choice for cervical cancer prevention. The American Society of Colposcopy and Cervical Pathology (ASCCP) recently released the Risk-Based Management Consensus Guidelines for Abnormal Cervical Cancer Screening Tests and Cancer Precursors , which aThe treatment of cervical dysplasia varies depending on the patient’s age, pregnancy status, the extent of cervical intraepithelial neoplasia, and the estimation of the patient’s risk of developing CIN 3+ based on a combination of medical results and history. In all nonpregnant patients, regardless of age, with a histopathological diagnosis of CIN 3, treatment is recommended, and observation is unacceptable (AII), with excisional treatment preferred to ablative treatment ,22. In nSurgical excisional therapy can be performed using the loop electrosurgical excision procedure (LEEP), cold knife biopsy, or laser cone biopsy. Multiple studies have tried to determine the method of choice, but no technique has been found to be superior in terms of treatment failure or treatment-associated morbidity . A meta-As HPV persistence is the leading and only modifiable factor affecting the risk of the progression of CIN lesions into high-grade cervical dysplasia and cancer, it has been proposed to conduct adjuvant vaccination in patients treated for high-grade cervical dysplasia. As no vaccine has been approved for therapeutic use in patients diagnosed with HSILs to date, attempts have been made to determine the use of HPV prophylactic vaccination in order to reduce recurrent HSILs and prevent cervical cancer. To date, numerous studies have been conducted, but only a few were prospective. In The mechanism of function of the HPV vaccine in patients already infected with HPV is not fully understood. The reduction in the recurrence rates of HPV infections and HPV-induced lesions may be due to the following: For patients who have not been previously vaccinated, the vaccination may act as primary prevention and protect against new HPV infections. Another mechanism may prevent the loss of immunological effectiveness against HPV reactivation/reinfection in patients who did not develop long-lasting immune protection after a previous infection .p = 0.006) and CIN 2+  after surgical treatment with adjuvant HPV vaccination compared with the unvaccinated group. The authors also noticed a nonsignificant reduction in HPV persistence among vaccinated patients. A different meta-analysis conducted by Jentschke et al. [Accumulating data show a potential role of HPV prophylactic vaccines in the adjuvant setting to surgical treatment in patients with HSILs; however, study results differ regarding vaccination efficacy. A recent meta-analysis by Di Donato et al. , conducte et al.  showed ae et al.  also conAll up-to-date prospective studies have demonstrated a positive effect of adjuvant HPV vaccination and revealed lower rates of CIN 1+ and CIN 2+ recurrence. The most extensive study to date was a prospective cohort study conducted by Sand et al. , which iPost hoc studies of PATRICIA , FUTURE Similar studies regarding secondary HPV vaccination have been conducted in patients suffering from other HPV-related lesions. Adjuvant HPV vaccination after surgical treatment was found to reduce the incidence of subsequent vaginal intraepithelial neoplasia (VaIN), vulvar intraepithelial neoplasia (VIN), and genital warts . StudiesEven though there is no level I evidence for adjuvant HPV vaccination use in high-risk patients with HPV infection and HPV-associated lesions, multidisciplinary consensus evidence-based guidelines were created in Spain . In accoThe protective effect of secondary HPV vaccination seems to differ depending on HPV patient’s status. The most significant protective effect was demonstrated for patient-targeted HPV vaccine genotypes 16 and 18, giving a risk reduction of 63% . HoweverNot only debatable is the use of the HPV vaccine in recurrent HPV, but also the timing of its administration. Persistent HPV infection seems to be associated with alternations in the local microenvironment and increased levels of proinflammatory cytokines ,49. The p = 0.047). The study evaluated 306 patients; however, patients with immunosuppression, multicentric HPV disease, a history of any HPV-associated cancer, and/or a diagnosis of invasive disease in the surgical specimen were excluded from the study. Moreover, the study did not specify the exact vaccination timing and only stated that “the first dose of the vaccine is provided immediately before or after the treatment according to the availability of the vaccine and the timing for HSIL treatment”. A meta-analysis revealed no significant effect of vaccination timing on the protective vaccination effect [Different studies have used different vaccination timings. However, in most of them, the time of vaccine administration was either before LEEP/conization or shortly after (up to 1 month after LEEP). The study conducted by Sand et al.  revealedn effect ; howevern effect . As HPV n effect . The incn effect  revealedIn a study conducted by Del Pino et al. , vaccineImmunocompromised patients, including patients with HIV, are more likely to have HPV infections and develop HPV-related lesions. In these patients, cervical HSILs are more likely to be caused by non-vaccine HPV types when compared to women without HIV . The risFirnhaber et al.  conducteSince the beginning of HPV vaccinations, more than 270 million doses of HPV vaccines have been distributed. Following the Global Advisory Committee on Vaccine Safety GACVS of the WHO, HPV vaccines were classified as extremely safe . The safCurrent HPV vaccines were engineered as primary preventive vaccines. Their function is to activate the patient’s humoral immunity and production of virus-neutralizing antibodies in order to prevent the viruses from entering host cells. The vaccines were found to be effective in protecting against persistent HPV infections and the formation of premalignant neoplasia lesions through the induction of neutralizing antibodies (IgG and IgA).Therapeutic vaccines are constructed differently from prophylactic vaccines, as they should stimulate the cell-mediated immunity of the acquired immune cells, especially CD8+ T cells, rather than neutralizing antibodies . They shThe development of target vaccines requires additional time for the collection of confirmatory data and the creation of regulatory approval. In addition, despite the recent advanced techniques, the vaccines may not live up to the patient’s expectations due to their delivery methods, poor coverage of HPV genotypes, and limited safety data. Moreover, the success of the clinical trials may depend on patient selection criteria and the use of biomarkers of cancer invasiveness and prognosis to optimize patient selection and maximize potential therapeutic outcomes .With the increasing evidence of a positive effect of secondary vaccination in patients with HPV-related lesions, there is a need for prospective studies evaluating the influence of prophylactic vaccines on relapsed and/or recurrent cervical intraepithelial neoplasia. Risk stratification and the selection of the response criteria of patients who would benefit the most are crucial for effective treatment and secondary prophylaxis. A better understanding of secondary vaccination, its timing, and its cost effectiveness are essential to developing international guidelines. Adjuvant HPV vaccination after surgical treatment may reduce the risk of recurrent cervical dysplasia.


Purpose: One of the most known sexually transmitted diseases is Condylomata acuminata (CA), a skin lesion occurring due to infection from Human Papilloma Virus (HPV). CA has a typical appearance of raised, skin-colored papules ranging in size from 1 mm to 5 mm. These lesions often form cauliflower-like plaques. Depending on the involved HPV-subtype (either high-risk or low-risk) and its malignant potential, these lesions are likely to lead to malignant transformation when specific HPV subtypes and other risk factors are present. Therefore, high clinical suspicion is required when examining the anal and perianal area. Methods: In this article, the authors aim to present the results of a five-year case series (2016–2021) of anal and perianal cases of CA. Results: A total of 35 patients were included in this study. Patients were categorized based on specific criteria, which included gender, sex preferences, and human immunodeficiency virus infection. All patients underwent proctoscopy and excision biopsies were obtained. Based on dysplasia grade patients were further categorized. The group of patients where high-dysplasia squamous cell carcinoma was present was initially treated with chemoradiotherapy. Abdominoperineal resection was necessary in five cases after local recurrence. Conclusions: CA remains a serious condition where several treatment options are available if detected early. Delay in diagnosis can lead to malignant transformation, often leaving abdominoperineal resection as the only option. Vaccination against HPV poses a key role in eliminating the transmission of the virus, and thus the prevalence of CA. Condylomata acuminata (CA), otherwise known as anogenital warts, are caused by human papilloma virus (HPV) infection. HPV is one of the commonest sexually transmitted diseases worldwide, with 9–13% of the world population affected , commonlHPV is a non-enveloped, double-stranded DNA virus from the papilloma virus family that infects the nucleus of differentiated squamous cells ,3. It caThe HPV genome consists of a transcribed region which encodes six early proteins, namely E1, E2, E4, E5, E6, and E7. Some of these HPV proteins are strongly type-specific and they can be detected in certain neoplastic lesions. Especially in the HR-HPV subtypes increased levels of E5, E6, and E7 oncoproteins seem to affect and activate multiple signaling pathways and thus stimulate proliferation of infected cells. Although life-cycle organization between HR- and LR-HPV subtypes is similar, the main difference lies in the strongly different ability of each group’s oncoproteins (mostly E6 and E7) to control cell cycle entry in the basal/parabasal cell layers ,9.HPV invades the cells of the epidermal basal layer through microabrasions . TransmiHigh-risk HPV subtypes are responsible for certain types of cancer of the cervix ), anus, penis, vagina, vulva, oropharynx and even esophageal adenocarcinoma (EAC). Subtypes HPV-16 and HPV-18 are held responsible for the majority of cervical cancers and almost 95% of HPV-positive oropharyngeal cancers (OPCs). Other types of high-risk HPV related neoplasia are the vulvar high-grade squamous intraepithelial lesions (vH-SIL) which can progress to invasive vulvar cancer. From the low-risk HPV subtypes, some persistent ones, such as HPV-6 and HPV-11, cause recurrent conditions and mostly involve anogenital warts, respiratory papillomatosis, and vulvar low-grade squamous intraepithelial lesions (vL-SIL), which are considered to represent a benign condition ,13.®9), the quadrivalent vaccine for types 6, 11, 16, and 18 (Gardasil®), and the bivalent vaccine for the high-risk types 16 and 18 (Cervarix®). In Greece, HPV vaccination is offered to girls 11–18 years old and boys and girls 11–26 with risk factors  .Herein we present a 5-year case series (2016–2021) of anal and perianal CA.The patients selected had surgical resection of anal and perianal warts between 1/1/2016 and 1/1/2021 (60 months) in the 3rd Surgical Department of University General Hospital of Thessaloniki AHEPA, by a single surgical team. Patient data were collected using the histopathological reports of the specimens resected, which were acquired from the Histopathology Laboratory of Aristotle University of Thessaloniki, along with the patient records from the 3rd Surgical Department’s archive. Inclusion criteria included excision of anal and perianal lesions and exclusion criteria included final histopathological results of CA with high grade, mild-moderate dysplasia, and SCC.A total of 35 patients were included in this study. The patient cohort consisted mostly of men: 32 patients were male (91.4%) and three were female (8.6%), with an average age of 39.9 years. The patients were between the age of 17 and 73 years. Moreover, 13 of the male patients (37.1%) reported to have sex with men (MSM) . All HIVAll patients had proctoscopy under general anesthesia and excision biopsy of all suspicious perianal and anal lesions . ExcisioHistopathology reports illustrate 16 patients with condylomata acuminata without dysplastic or neoplastic features (45.7%). Five patients had mid- to high grade dysplasia (14.3%); four patients had high grade dysplasia (11.4%); 10 patients (28.6%) were found to have invasive squamous cell carcinoma (SCC) in their histopathology results; two patients had Buschke-Lowenstein tumors (5.7%) .The remaining 25 patients whose histopathology results showed no presence of confirmed malignancy did not receive any further treatment. Excision of the lesions was considered enough. These patients continue to have annual follow ups with proctoscopy and remain disease-free.Anal and perianal condylomata acuminata represent a controversial subject, largely due to limited clinical trials and high-level evidence, especially in terms of screening and prevention. There are currently no official guidelines regarding large scale vaccination for males and screening for anal SCC or HSIL . Although gender distribution appears to be roughly equal in the literature, the vast majority of the patients in this cohort were male (91.4%). This could be due to a number of factors. Nowadays, most women have regular screening for cervical cancer and consult their gynecologist frequently, who may manage a significant number of perianal warts. This department has a close cooperation with the HIV Medicine Unit of Aristotle University of Thessaloniki, which could account for the increased male predominance as well as the high percentage of HIV positive patients in this cohort.Another significant observation to note is the large percentage of HSIL and invasive SCC. Interestingly, only a 2–4% rate of malignant transformation is reported in the literature, whereas we found 28.6% of our patient cohort had invasive SCC and nine patients (25.7%) had mid- to high grade dysplasia. HIV infection could, of course, contribute to the notably high percentage of dysplasia and invasive SCC, as it has been shown to be a risk factor for malignant transformation of CA ,18,19. TPatient information and prevention remains poor. Current HPV vaccination guidelines in Greece include girls from 11 to 18 years old as well as boys and girls from 11 to 26 years old in high-risk groups  ,23. The The biggest limitation of our study is the small sample size. However, the high percentage of HIV positive patients and the significant percentage of HSIL and SCC in our sample highlights the importance of regular follow up, as well as screening, especially in high-risk patients. It also illustrates the gap in preventive strategies and immunization in the male population. The need for further studies and high-level evidence in the management of CA and especially in the prevention of malignant transformation is indisputable.Another important element regarding management of CA is high clinical suspicion. Physicians must be aware of the prevalence of this condition, the age-groups and other population sub-groups that are mostly affected, and its possible clinical manifestations. This verrucous hyperplasia can often be misdiagnosed due to its clinical presentation as multiple growths on the skin or mucous membranes . They caFinal diagnosis, however, is set only after excision of the lesions through biopsy and histopathological examination. Serology tools and methodologies, including polymerase chain reaction (PCR) detection and DNA detection assays, are not considered to be reliable with an estimated level of evidence of 2b. Moreover, it is not possible to have cultures of the virus. While the sample that can be used to detect the virus using PCR can be the same as the one used for cytological examination, several limitations are present regarding these methods. Most of the time, only a small sample is obtained, which can lead to sampling errors even when very sensitive assays are used. Another limitation concerns the menstrual cycle, which can also tamper with serology and molecular results ,28. MoleMost HPV infections are subclinical and are usually handled and resolved by the immune system. HPV types can be further subcategorized to mucosal and cutaneous types. Mucosal types affect mucous membranes causing anogenital warts and neoplasia of the cervix. The squamous epithelium of the skin is infected by the cutaneous types, causing all forms of common warts, which can lead to CA. When specific oncogenic types of HPV are involved, the infected patient may develop cervical, oropharyngeal, anal, vulvar, vaginal cancer, and cancer of the penis ,7,30.While malignant potential and evolution to cancer strongly depends on the HPV type there are other risk factors that make certain patients more prone to an invasive type of cancer. These co-factors include mainly smoking, which seems to increase the risk of progression to high-grade dysplasia of the existing lesions, and presence of HIV infection. Immunosuppression caused by HIV significantly affects the behavior of HPV-related tumors as well as the outcome of those patients, findings that were confirmed also by the results of our cohort. The expected clearance of HPV from the immune system is significantly reduced in patients with HIV. Therefore HIV-positive patients develop HPV-related malignancies such as CA which tend to be more aggressive at a younger age and at an advanced stage upon diagnosis compared to HIV-negative patients . Another2), argon plasma coagulation, cryotherapy, local hyperthermia, and some medications [The optimal management of CA varies depending on the lesions’ size and location, but it usually includes surgical excision combined with another non-invasive technique . These tications ,33,34,35ications ,37. Injeications ,39. Seveications . In caseWhile effective in removing the skin lesions, these treatment options do not offer a chance to eradicate HPV from the human body. While macroscopical lesions can be removed effectively and often without leaving any invaded margins, HPV cannot be cleared out from the human body. This leads to the realization that it is possible to have relapses and reoccurrence of HPV-related lesions whenever certain risk factors are present and/or the immune system appears more vulnerable. Important research has been conducted in order for immunotherapy to be established as a high-efficacy treatment option. Towards this direction, Mastutik et. al. studied protein p16INK4A expression, which seems to be highly related to HR-HPV infection and may serve as a biomarker for the prediction of malignancy potential in CA lesions .While the eradication of HPV infection and HPV-related lesions especially in individuals with risk factors is far from available, the need for prevention must be underlined. Primary prevention through immunization is already applicable to young girls aged 10 years  and older requiring two or three vaccination doses based on the age of first vaccination, although most national vaccination guidelines suggest a three-dose immunization scheme ,41. The HPV remains one of the most frequently encountered STIs worldwide. Although important progress has been made in terms of preventing virus transmission through vaccine immunization, the virus remains untraceable in many cases. CA, as the clinical manifestation of HPV, affects a notable percentage of the population, especially young people. At this point, high clinical suspicion is required from all clinical doctors as CA has more treatment options and better prognosis if treated early. A delay in diagnosis can lead to malignant transformation, often leaving abdominoperineal resection as the only option. This study, although small in terms of the sample considered, aims to shed light on CA’s prevalence and patients’ special characteristics in a European country where not much data on the matter are available at the moment. Nevertheless, more studies are required in order to fully understand and target the most vulnerable population subgroups, raise awareness, and generalize the prevention of CA through immunization.


Monitoring HPV vaccines post-licensure plays an important role in assessing the progress of immunization programs, demonstrating the impact of vaccines on the population, and providing data for policy needs. In Colombia, HPV vaccines showed effectiveness when administered before start of sexual activity, and two doses are sufficient to achieve good protection P < 0.001), whereas for HPV6/11 infections, a decrease of 63.7% in vaccinated women (1.02% vs. 2.81%) was observed. The adjusted effectiveness to HPV16/18 was 61.4%; 95% CI, 54.3%–67.6%. However, the effectiveness against HPV16/18 was significantly higher among women vaccinated before their sexual debut 91.5%; 95% CI, 86.8–94.5, compared with effectiveness for vaccination after their sexual debut, 36.2%; 95% CI, 23.6–46.7. Five years after the introduction of HPV vaccines in Colombia, high effectiveness of HPV to prevent HPV16/18 infections is observed in the catch-up cohorts including virgin and sexually active women.In Colombia, the human papillomavirus (HPV) vaccine was launched in 2012 in the context of a school-based national vaccination program targeting girls ages 9 to 14 and offering catch-up vaccination for girls ages 14 to 17. In this study, we evaluated the program's impact on type-specific HPV infection by comparing HPV cervical prevalence among vaccinated and nonvaccinated women. This is a comparative cross-sectional study conducted 5 years after the quadrivalent HPV vaccination implementation in a sentinel Colombian City. This study included young women (18–25 years old) who had been vaccinated in the catch-up group and were attending universities and technical institutions, and women who attended primary health care facilities for Pap smear screening. The HPV prevalence of 1,287 unvaccinated women was compared with the prevalence of 1,986 vaccinated women. The prevalence of HPV16/18 infections was significantly lower in vaccinated compared with unvaccinated women (6.5% vs. 15.4%; Monitoring HPV vaccines post-licensure plays an important role in assessing the progress of immunization programs, demonstrating the impact of vaccines on the population, and providing data for policy needs. In Colombia, HPV vaccines showed effectiveness when administered before start of sexual activity, and two doses are sufficient to achieve good protection. National human papillomavirus (HPV) vaccination programs have been implemented in many countries with coverage ranging from 20% to 70% . Even thFrom 2013 onward, the target age was expanded to 9 to 17 years old (catch-up group) and the original immunization schedule was modified to an alternative one . Data were captured online in Redcap.Cervical smears from unvaccinated women were collected between May 2014 and February 2015 as part of a baseline group for further comparisons , and smeThereafter, each woman underwent a gynecologic examination, and two cervical samples (one each for Pap and HPV testing) were obtained as it was previously published . The cytDNA extraction was performed using the QIA-cube with the AmpliLute Liquid Media Extraction kit, following the manufacturer's instructions (Roche Diagnostics), and subsequently, the HPV detection and genotyping were assessed with the Linear Array HPV Genotyping Test (Roche Diagnostics), which detects 13 high- and 23 low-risk HPV types, as it was previously published .The HPV type–specific prevalence was estimated as the proportion of participants who tested positive for a given HPV type. Prevalence for unvaccinated and vaccinated women were estimated as follows: any HPV type, any high-risk HPV type (HR-HPV); any low-risk HPV-type (LR-HPV); nonvaccine HR-HPV types, HPV6–11–16–18, HPV16–18, HPV6–11, and HPV31 and 45, as there is some evidence of cross-protection on them.Given the differences between unvaccinated and vaccinated women, a propensity score matching based on inverse probability of treatment weighting was performed to balance differences in covariates between the two groups . The covComparisons in demographic characteristics, HPV prevalence rates, and cytological results between the two groups were performed using the chi-square test for categorical data. The propensity score was used to balance differences in baseline covariates between unvaccinated and vaccinated women in total and stratified by vaccination status. Statistical analysis was performed using R-project v4.0.0 . The covariate balance for the propensity score matching was done with the cobalt package in R .Cytological results were stratified according to the 2014 Bethesda System (TBS). Comparisons of HPV types prevalence according to Pap smear results between the groups were performed. An ordinal logistic regression model was used to adjust prevalence estimates after propensity score matching .P < 0.05.Vaccine effectiveness was estimated as 100 × [1 − odds ratio] with the corresponding 95% confidence intervals (95% CI). Effectiveness was also estimated according to sexual debut and the number of vaccine doses. The level of significance was A total of 3,465 women ages 18 to 25 years were invited, and 3,273 were included in the study: 1,426 of them were unvaccinated (41.15%) and 2,039 vaccinated (58.84%). Of the 1,426 nonvaccinated women, 139 were excluded due to samples not suitable for HPV typing (59 women), B-globin negative (38 women), and no cytology report (42 women). Among vaccinated women, 53 were excluded because they did not meet the inclusion criteria. The final population for analysis corresponds to 1,287 (39.3%) unvaccinated and 1986 (60.7%) vaccinated women.Among vaccinated women, 33.4%  received at least one dose of vaccine, 63.9%  received two doses, and only 2.7%  went on to complete the full three-dose vaccination schedule. 53.5%  of them had received their first dose before sexual debut.P values after propensity score adjustment were all >0.05, and the standardized differences for most of the variables were <20%, which indicates that variables were successfully balanced (The characteristics of the study population are described in P = 0.31). Likewise, no significant differences in prevalence of non-HPV16–18 between unvaccinated and vaccinated women , and non 6–11 HPV types  were observed (P = 0.24). However, a significant increase in HPV62 , HPV84 , HPV42 , and HPV67  was observed in vaccinated women .After balancing by using the propensity score, no significant differences in prevalence of any HPV were observed between unvaccinated and vaccinated women , while for HPV18 it dropped from 5.45% to 2.09% . As for HPV6, a slight decrease was observed (from 1.86% to 1.02%), though it was not significant, P = 0.06; Supplementary Fig. S2; Supplementary Table S2). Overall, a reduction of 63.5% for all vaccine types, from 17.6% to 7.4%, was observed  and 5.03% vs. 4.71% (P = 0.66), respectively; P = 0.97; Regarding related HR-HPV types, no significant differences in prevalence for HPV31 and HPV45 in unvaccinated versus vaccinated women were observed [4.13% vs. 3.94% (P < 0.00). Prevalence of HPV vaccine types with two doses decreased by 83.06% in vaccinated women, from 17.6% to 2.98% (P < 0.001). Similar changes in HPV prevalence rates were observed for HPV16–18 , with a reduction of 85.5%. In contrast, with one dose, the observed reduction was only 25% for HPV vaccine types (17.6%–13.2%), and 21.4% for HPV 16/18 .As far as doses were concerned, after balancing by using the propensity score, complete protection to HPV vaccine types was observed with three doses , P > 0.05, neither against HR-HPV≠16/18: −11.10, 95% CI, −22.9 to −0.4, P > 0.05. Similarly, no vaccine effectiveness against HPV31 and 45 was observed: 7.2%, 95% CI, −28.1 to 10.4, P > 0.05 , compared with those after sexual debut , being the highest after two doses . However, considering severity of cervical lesion as a clinical outcome, using ordinal logistic regression, no significant reduction was observed in the risk of cervical lesion .Compared with unvaccinated women, the percentage of normal cytology was significantly higher in vaccinated women (90.5% vs. 93.7%), respectively, P < 0.001). Similar results were observed for HPV18 . No differences in the prevalence of ASC-US among unvaccinated and vaccinated women were observed . The proportion of ASC-US attributable to HPV16 was lower in vaccinated women (9.73%) compared with unvaccinated women (15.3%), although it was not significant (P = 0.68). No differences for HPV18 were observed between two groups. However, a significant reduction of ASC-US attributable to HPV45 in vaccinated compared with unvaccinated women was observed . In contrast, a significant increase of ASC-US associated with HPV39 and 51 was observed . However, no infection associated with HPV 16 in vaccinated women  was observed. Similarly, a lower proportion of L-SIL attributable to HPV18, 31, 45, and 39 was observed, but it was nonsignificant , though it was not significant , Monitoring HPV vaccines post-licensure plays an important role in assessing the progress of immunization programs, demonstrating the impact of vaccines on the population, and providing data for policy needs . This isIn line with other surveillance studies, which have reported an important reduction in prevalence for HPV6, 11, 16, and 18 in other populations, our study showed a substantial reduction in the prevalence of HPV vaccine types during the first 5 years after vaccine introduction in sexually active young women, providing evidence of direct protection by the quadrivalent vaccine, and suggesting high vaccine effectiveness in a real-world setting. In similar studies, a reduction of HPV16/18 from 19.1% to 6.5% among 16- to 18-year-old women (66% reduction) was reported in Great Britain . In ScotIn our study, even though complete protection against HPV16/18 infections was observed in young women who received three doses of the HPV vaccine, we found that women who received two doses of vaccine had higher effectiveness (93.2) against HPV vaccine-type infection as compared with those who had one dose (84.9). These findings confirm the high effectiveness suggested by other studies, meaning that at least two doses are needed to achieve a high level of immunity across time and that it is still highly effective when given at older ages. Nevertheless, it has been found that young girls may have a better immunologic response to the HPV vaccine . Thus, tCompared with the bivalent vaccine, the quadrivalent vaccine shows a more limited cross-protection, but a significant efficacy has been noted, mostly against HPV31 . In thisFollowing a reduction in HPV vaccine-targeted genotypes, an increase in infection or disease due to other HPV types, not prevented by the vaccine, may occur because of reduced competition during natural infection . HoweverIn a previous study, Gray and colleagues reported a sporadic HPV39 and 51 occurrence, but no patterns suggestive of type replacement following vaccination with the bivalent HPV16/18 vaccine.In the same way, in this study, we observed a significant increase in ASC-US associated with HPV39 and HPV51 infections; however, this increase was not observed in L-SIL. These results confirm that, although no conclusive signs of type replacement are observed, it is important to continue monitoring the incidence of HPV39 and HPV51 in future investigations . Lastly,HPV16 and 18 types have been detected in about 34% of L-SIL in cytological abnormalities, reflecting that these low-grade lesions could indicate acute HPV infection . In our The greatest impact of the HPV vaccine is expected to happen in low-income and middle-income countries in which there is no screening or only limited screening for cervical cancer and where the highest burden of cervical disease is generally observed. The impact of vaccination with current HPV vaccines in low resource settings, without any other preventive actions, is estimated to potentially reduce cancer risk by 40% to 50% with 70% vaccination coverage. The result of this study is an early indication that the national HPV program has been successfully preventing HPV16/18 infection in sexually active women, and is promising in terms of its implications for declining rates of CIN, and ultimately of ICC in the future. However, despite the successful uptake of the vaccine, the program was adversely affected by controversy and misinformation regarding its safety, and coverage has been in decline, specifically after the event of “El Carmen de Bolivar” . We hopeOne of the strategies used to monitor the early impact of the HPV immunization program is the use of residual specimens from sexually active young women undergoing chlamydia screening . Yet, thThis study also has some limitations that should be interpreted with caution when addressed. First, this is a cross-sectional study carried out in two different periods, and even though strict comparability of samples between two periods is a general weakness of nonrandomized comparative studies, in this study, we collected the unvaccinated and vaccinated samples in the same geographic locations and in the same institutions. In an attempt to minimize differences in HPV risk-related characteristics between the two sample periods, we have adjusted all the available variables for comparing the two groups by using a propensity score analysis based on inverse probability of treatment weighting. We found that the decrease in HPV prevalence was maintained after adjusting the differences between the samples in demographic and other features. However, it is possible that differences in HPV prevalence may have been attributable to factors other than those measured in this study . On the In conclusion, this study provides a preliminary evaluation of the vaccine effectiveness against vaccine-targeted HR-HPV type infections following the implementation of the national HPV vaccination program in Colombia. In line with international data, five years after the vaccine was introduced in Colombia, we have shown a substantial and statistically significant decrease in the prevalence of vaccine HPV genotype infections. We did not find indications of cross-protection against HPV31/45. However, due to coverage decline in 2015, it is necessary to improve coverage rates among women and continuously monitor the vaccine impact, to evaluate if this decrease results in preventing preneoplastic lesions and invasive cervical cancer over time.No disclosures were reported.