Rhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines worldwide and it is responsible for the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Aquaporins (AQPs) are water channel proteins implicated in physiological mechanisms of osmoregulation. Members of the AQP family are critical for blood-feeding arthropods considering the extreme osmoregulatory changes that occur during their feeding. We investigated the pattern of expression of a newly identified AQP2 gene of R. microplus (RmAQP2) in different tick tissues and stages. We also examined in vivo the biological implications of silencing expression of RmAQP2 silencing during tick feeding on either uninfected or B. bovis-infected cattle.Ticks are blood-feeding arthropods that can affect human and animal health both directly by blood-feeding and indirectly by transmitting pathogens. The cattle tick In silico gene analyses were performed by multiple alignments of amino acid sequences and topology prediction. Levels of RmAQP2 transcripts in different tick tissues and stages were analyzed by reverse transcriptase quantitative PCR. Patterns of expression of RmAQP2 protein were investigated by immunoblots. Gene silencing was performed by RNA interference and in vivo functional analyses carried out by feeding ticks on either uninfected or B. bovis-infected cattle.RmAQP2 transcripts were found in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged females; however, of all tick tissues and stages examined, RmAQP2 protein was found only in salivary glands of partially engorged females. RmAQP2 silencing significantly reduced tick fitness and completely abrogated protein expression. The effect of RmAQP2 silencing on fitness was more pronounced in females fed on a B. bovis-infected calf than in ticks fed on an uninfected calf and none of their larval progeny survived.RmAQP2 is critical for tick blood feeding and may be a suitable candidate target for the development of novel strategies to control R. microplus and tick-borne parasites.Collectively, considering the gene expression and tick fitness data, we conclude that The online version of this article (doi:10.1186/s13071-015-1226-2) contains supplementary material, which is available to authorized users. Aquaporins (AQPs) are a family of transmembrane proteins that form pores to transport water and small solutes across cellular membranes . MembersNumerous members of the AQP family have been identified in arthropods in the last few years due to the availability of several arthropod genomes and genetic information, such as transcriptomes and cDNA libraries . StudiesRhipicephalus (Boophilus) microplus is one of the most economically important ectoparasites of bovines, as it is the main vector of the apicomplexan protozoan Babesia bovis, the etiological agent of bovine babesiosis , which was used to obtain the RmAQP2 sequence, and confirming that our laboratory colony represents a prototypical strain of R. microplus , Dermacentor variabilis AQP9 [GenBank ID: ABI53034.1], R. sanguineus AQP1 [GenBank ID: CAR66115.1], Ixodes ricinus AQP1 [GenBank ID: CAX48964.1], I. scapularis AQP1 [GenBank ID: XP_002399532.1] and I. scapularis AQP2 [GenBank ID: XP_002400655.1], respectively  , engorged nymphs (10 nymphs per sample), unfed males , and individual salivary glands, ovaries and guts of partially engorged females (at day 5 of feeding). We analyzed six biological replicates of each sample by RT-qPCR. Relative levels of expression of RmAQP2 were different in larvae (2.5 [±0.66]), nymphs (3.0 [±0.23]), female guts (3.3 [±0.99]) and female salivary glands (9.5 [±1.73])  than in larvae, nymphs and guts of females.The transcription level of pression . TherefoRmAQP2 mRNA in unfed larvae, engorged nymphs, and guts and salivary glands of partially engorged females, we investigated the presence of RmAQP2 protein in these tick stages and tissues . Both dsRNA fragments were approximately 400 bp in length and their respective location on the RmAQP2 gene is represented in Additional file RmAQP2 silencing were obtained when either one or two dsRNA fragments were used for silencing. RmAQP2 was silenced 92.8 % (±0.26 %) and 99.3 % (±3.70 %) in ticks injected with either one dsRNA or the two non-overlapping dsRNA fragments, respectively   . In both experiments after the dsRNA-injected females were fed to repletion, the percentage of engorged females, weight of engorged females, oviposition rate, egg masses, percentage of hatching and percentage of larvae survival were evaluated (Tables B. bovis infection (experiment 1), identical numbers of females from both, the RmAQP2 silenced group and control group, fed to repletion. The average weight of the RmAQP2-silenced engorged females was 361.8 mg and significantly higher (P < 0.05) than the control group (342.4 mg). Silencing of RmAQP2 had no effect on the percentage of oviposition and egg masses between the two groups of ticks. In contrast, only 50 % of the egg masses hatched compared to 90.2 % hatching of the control group. Strikingly, none of the larvae survived in the RmAQP2-silenced group compared to 100 % larvae survival in the control group  than the average weight of engorged females in the control group (305.6 mg). Oviposition rate was not affected by RmAQP2 silencing; however, egg mass weight was significantly lower (P < 0.05) in the silenced group (134.7 mg) than in the control group (203.4 mg). Interestingly, the percentage of hatching was significantly reduced (P < 0.05) in the silenced group (81.2 %) compared to the control group (97.7 %); however, 100 % of the larvae survived in both the silenced and control groups (Table After removal of the ticks for assessment of silencing and protein expression, the reminder of the ticks in the two independent experiments were allowed to feed to repletion to investigate the R. microplus ticks. RmAQP2 transcript was demonstrated in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged female ticks. Interestingly, however, among the ticks tissues and stages investigated, RmAQP2 protein was found only differentially expressed in the salivary glands of partially engorged females. In vivo functional analyses showed that silencing of RmAQP2 decreased tick fitness and this effect was more pronounced in ticks fed on a calf during acute B. bovis infection. Noteworthy, none of the larval progeny from the RmAQP2-silenced females that were fed on a B. bovis-infected calf survived. Considering that R. microplus larvae transmit B. bovis, these data may have implications for the control of tick-borne parasites [In the present study we investigated the pattern of expression of a newly identified aquaporin 2 of arasites .R. microplus is a one-host tick that infests the host as larvae and remains until the adult females have fed to repletion, we did not think it was meaningful to assess all tick life stages of the tick for aquaporin expression. The adult female takes the majority of the blood. It is that stage that is primarily responsible for acquisition of B. bovis and it is also that stage that causes the most direct damage to the host by feeding. B. bovis is passed from the adult female transovarially to the larval offspring and it is the larval stage that transmits B. bovis. Consequently, the most biologically relevant stages of the tick for acquisition and transmission of B. bovis are the adult female and the larvae, respectively. Adult females are the most biologically relevant stages with regard to damage cause by tick feeding. Therefore, our investigations focused primarily on these two stages for expression of the aquaporin gene.Since D. variabilis, R. sanguineus, I. ricinus, I. scapularis and I. scapularis [R. microplus ticks. The presence of two NPA domains and the level of amino acid identity between RmAQP2 and other AQPs prompt us to argue about a potential evolutionary conserved function for these proteins. The remarkable similarity of topology among AQP members in different species, including RmAQP2 described in this study, also supports the prediction of a putative conserved function [R. microplus infestation and R. microplus-borne parasites. It has been proposed that AQPs play a critical osmoregulatory function in blood-feeding arthropods during saliva production, intake and excretion of water during blood digestion, reproduction and egg development, and off-host environment stress tolerance [RmAQP2-silenced female ticks had a significant increase in body weight at the time of repletion compared to control non-silenced ticks, suggesting a level of involvement of RmAQP2 in osmoregulatory mechanisms. Additionally, the increased body weight implies that ticks accumulate more liquids in the absence of RmAQP2. Despite being heavier, the RmAQP2-silenced group produced egg masses of reduced weight and reduced hatching success when compared with the control group, suggesting a negative correlation between intake of blood and generation of viable larval offspring.Members of the AQP family have recently been identified in several arthropods including tick species, such as apularis , 29. Herfunction , 2, 4. Afunction . This maolerance . SalivarRmAQP2 interfered with the osmoregulatoty system of R. microplus during feeding as demonstrated by the increased body weight in the gene-silenced engorged females. Effect on tick fitness was even more pronounced in ticks fed on a B. bovis-infected calf perhaps due to the deleterious presence of the apicomplexa parasite infection [RmAQP2 silencing throughout the tick life cycle considering that none of the larval progeny survived from the silenced females that fed on the calf during acute B. bovis infection. Here we also show that dsRNA injection abolished the expression of RmAQP2 protein completely in salivary gland of partially engorged females at 5 days of feeding. This result suggests a possible rapid turnover of RmAQP2 in salivary glands. Rapid turnover of AQPs has been previously demonstrated both in animal and plant tissues and shown to be more pronounced during infections or when organisms are under stress [AQP1 mRNA of I. ricinus ticks is expressed only in female tissues involved in water flux, such as guts, rectal sac and especially abundant in salivary glands [RmAQP2 transcripts. However, RmAQP2 protein was found in salivary glands. Therefore, it is plausible to assume the presence of a mechanism to suppress translation of RmAQP2 in female guts, larvae and nymphs. Silencing of I. ricinus AQP1 affected feeding performance, but the ticks remained feeding on the host with subsequent potential for pathogen transmission. Therefore, the authors concluded that I. ricinus AQP1 is not a suitable target to control tick infestation or block transmission of tick-borne diseases [RmAQP2-silenced female ticks ingested more blood and consequently showed increased engorgement body weight. Hatching and larvae survival were also affected by gene silencing and most importantly, none of the larval progeny from the RmAQP2-silenced females fed on a B. bovis-infected calf survived. Differences in site and level of expression, and permeability may explain the discrepancies between the present study and the previous published data regarding I. ricinus AQP1 [R. microplus as an anti-tick vaccine [R. microplus AQP1 effectively reduced the number of adult female ticks that fed to repletion.Considering that RmAQP2 protein was only found in salivary glands, it is reasonable to assume that this AQP plays an essential role in osmoregulation during blood feeding and digestion. The data showed that knockdown of nfection . Additior stress –32. The y glands . Here wediseases . Our datnus AQP1 . Another vaccine . The autin silico analysis using NetOGlyc [RmAQP2 cDNA sequence obtained from the R. microplus gene index project may be incomplete at the 5′ end. This sequence was derived from an EST project and although it has two stop codons in the 3′ end of the sequence, a stop codon in-frame with the presumed start ATG codon is not present. Nevertheless, this possibility is unlikely considering that the topology analyses revealed a typical predicted conformation for the AQP family [R. microplus.Based on the amino acid sequence, RmAQP2 has a predicted molecular weight of ≈ 30 KDa. However, preliminary NetOGlyc  and NetPNetOGlyc  revealedNetOGlyc –37 and iP family , 28. RegR. microplus genome sequence is not available and the alignment analyses are restricted to sequences listed in databases. Blast analysis of the dsRNA sequences used in this study did not reveal significant homology to any known tick sequence other than RmAQP2. Additionally, gene silencing was validated by RT-qPCR and RmAQP2 was silenced at similar levels when we used either one or two different segments of dsRNA identical to the target gene. Taken together, these aspects support the data and represent the best possible strategy to make solid scientific observations regarding the biological effect of RmAQP2 silencing in R. microplus.RNA interference has been widely used to investigate gene function in arthropods. However, off-target effects have been described in some species  and cannR. microplus ticks. RmAQP2 transcripts were present in unfed larvae, engorged nymphs, and salivary glands and guts of partially engorged female ticks. However, among the distinct adult tissues and life stages tested, RmAQP2 protein was only found in salivary glands of partially engorged females. In vivo investigation demonstrated that silencing of RmAQP2 decreased tick fitness. The effect of RmAQP2 silencing on tick fitness was even more pronounced in females fed on a B. bovis-infected calf and none of their larval progeny survived. Therefore, we conclude that RmAQP2 is involved in osmoregulation during feeding and is a potential target for the development of novel strategies for the control of R. microplus and R. microplus-borne diseases.Here we demonstrate the pattern of expression of a newly identified aquaporin 2 gene of


To investigate the distribution of Human papillomavirus (HPV)-31 A, B and C variants as well as the common amino acid polymorphisms in Chinese women, all 14 HPV-31 positive cervical exfoliated cell specimens identified from a descriptive study including ∼2700 women from Northern China were analyzed. HPV-31 positive specimens were identified by Mass Spectrometry and the fragments of partial Long Control Region, E6 and E7 were amplified and directly sequenced or cloned into vector and then sequenced to confirm the variant information. HPV-31 prevalence in Northern Chinese female population was 0.52%. Six different sequences represented all 14 isolates, and these isolates were subsequently classified into variant lineage A (9), B (0) and C (5) by phylogenetic analysis. Five common amino acid polymorphism sites (2 in E6 and 3 in E7) and a novel non-synonymous mutation were detected in the current study. Our investigation suggested that HPV-31 was much less detected in Chinese women population than that in western countries. A and C variants were commonly detected while B variants were rarely detected in this population. Human papillomavirus (HPV) is a family of double strand DNA viruses. The classification of HPV is based on the conserved L1 sequence coding for the major capsid protein P = 0.05), but this trend was not observed in Caucasian women (P = 0.85) P<0.001), respectively HPV-31 is one of the high risk types most closely related to HPV-16. To date, studies have shown many intratypic variations in the region of L1, E6, E7 and Long Control Region (LCR) of HPV-31 worldwide To date, studies on cervical infection of HPV-31 variants have mainly been conducted in western countries, and little information has been provided in Asian countries, especially China. In this study, cervical exfoliated cell specimens collected from 2700 women in Northern China were tested, and the distribution of HPV-31 variant lineages and amino acid polymorphism was described.In 2007–2009, a population-based esophageal cancer cohort study was initiated in 9 villages in rural Anyang, China Research protocols and materials of this study were approved by the Institutional Review Board of the Peking University School of Oncology, China. All participants in this study provided written informed consent.Exfoliated cells from the cervix were collected by experienced gynecologists using saline-soaked swabs and cells were rinsed into an eppendorf tube filled with saline. All specimens were then centrifuged at 5000 rpm for 5 min and supernatants were discarded. All specimens were stored at −20°C and subsequently transported to our laboratory in Beijing and stored in ultralow temperature freezers (−70°C) until the DNA extraction and HPV detection.A questionnaire was completed by all participants during the interview to obtain the demographic data and personal information including age, place of residence, tobacco use history, together with the number of sexual partner and pregnancy history etc.5′-TGTTTAAACTGCCAAGGTTGTG, the reverse external primer: 5′-CATAAAACCAACCATTGCATCC, the forward internal primer: 5′-TGGAACAACATTAGAAAAATTGACA, the reverse internal primer: 5′-TCTTCTGGACACAACGGTCTTT. PCR was performed using Phusion High Fidelity Taq Polymerase . The kit ABI PRISM BigDye Terminator Cycle Sequencing v 3.1 was used in the sequencing procedure. Sequences obtained were checked for quality using chromas software. Blast tool of NCBI was used to identify novel sequences. A viral isolate was defined as a novel variant if as compared with other isolates, there was one or more nucleotide variations in the region analyzed DNA was extracted using Biomek 3000 system  and then tested using Mass Spectrometry (MS) to detect infections of HPV 68, 66, 59, 58, 56, 52, 51, 45, 39, 35, 33, 31, 18, 16, 11 and 6  Duplicated PCR was performed in order to investigate the reproducibility of our method. A hundred percent identical sequences were observed from duplicated PCRs (data not shown).Multiple sequence alignments of the partial LCR, E6 and E7 ORFs and distance matrix bootstrap analyses were performed using Clustal-X program Among the 2703 specimens, 14  were HPV-31 positive . AnalysiFive novel isolates were identified from 14 HPV-31 positive specimens . Phylogenetic analysis based on Neighborhood Joining method was performed including all unique isolates in the current study (N = 6), accompanied by 19 sequences which had been reported in whole genome study Comparing with the prototype isolate, seven nucleotide alternations in the 3′ part of LCR,seven in E6, and six in E7 were identified and listed in In this descriptive study, we found that the prevalence of HPV-31 in northern Chinese women was 0.52% (95%CI: 0.28%–0.87%). HPV-31 variants in our sample could be classified into 2 clades, representing A and C variant lineages. The A variants were the most commonly detected, followed by C variants, and no B variant was detected. Five identified (2 in E6 and 3 in E7) amino acid polymorphisms and one novel amino acid polymorphism in E7 were found in this population. To our knowledge, this was the first study focusing on HPV-31 variants in Chinese women and the population-based specimens gained representation for general population as compared to hospital-based studies.Race-specific preferences of variant infection and discrepancies in viral properties have been identified and illustrated systematically in HPV-16. Phylogenetic analysis concerning viral sequences revealed that variants segregate based on the geographical origin and can be classified as European, African, Asian, Asian-American and North American For HPV-31 variants, phylogenetic analysis did not show any racial or geographic clustering worldwide. This phenomenon was also observed in HPV-33, 35, 52, 58 and 67, which is different from that in HPV-16 and 18 In the current study, the HPV-31 prevalence observed was 0.52%, which was similar to the prevalence (0.4%) reported by meta-analysis in women with normal cytology from the Asia region P = 0.004). However, the difference was not significant between Chinese women and Caucasian women (P = 0.221). This was an evidence to support the potential racial and geographic variation in HPV-31 variants distribution though phylogenetic analysis of HPV-31 isolates did not reveal ethnic clustering as observed previously for HPV-16 and 18. Besides Xi's study, another two hospital-based studies have also investigated cervical HPV-31 infections at the variant level. Chagas et al in brazil enrolled 35 HPV-31 positive specimens from women with abnormal cytology, and reported the proportion of A, B and C variants were 57.2%, 5.7% and 37.1%,respectively The current investigation only found variant lineage A (64.3%) and C (35.7%) in 2703 Chinese women which was notably different from Xi's study which reported the proportion of A, B and C variants were 41.7%, 21.1% and 37.2% in US female population Although epidemiological studies have shown the discrepancies of viral properties among HPV-31 variant lineages, it is still crucial to identify amino acid polymorphisms accounting for the difference, especially in E6 and E7 regions due to these oncoproteins are essential for the occurrence, development and maintenance of malignancy in cervical epithelial cells In this descriptive study, we found that the prevalence of HPV-31 in Northern Chinese women was 0.52% (95%CI: 0.28%–0.87%). The variant lineage A (9/14) of HPV-31 was the most commonly detected variants, followed by C variants (5/14), which was notably different from those in some western ethnic populations. Five frequent non-synonymous mutations among HPV-31 variant lineages in E6 and E7 protein  and a novel non-synonymous mutation (N93S in E7) were detected. This study provides important basic data for the subsequent association studies and related molecular mechanism studies on HPV-31 variants and cervical lesions in Chinese women.


The objective of this study was to compare the accuracies of double staining for p16/Ki-67 and the molecular test for high-risk HPV (hr-HPV) to identify high-grade cervical intraepithelial neoplasia (CIN2/CIN3) in women with cervical cytology of atypical squamous cells of undetermined significance (ASC-US) and low-grade squamous intraepithelial lesion (LSIL). Data were collected from 201 women who underwent cervical cytology screening in the Barretos Cancer Hospital and their results were categorized as ASC-US (n=96) or LSIL (n=105). All patients underwent colposcopy with or without cervical biopsy for diagnosis of CIN2/CIN3. The hr-HPV test (Cobas 4800 test) and immunocytochemistry were performed to detect biomarkers p16/Ki-67 (CINtec PLUS test). Two samples (1 ASC-US/1 LSIL) were excluded from the analysis due to inconclusive results of the histologic examination. There were 8 cases of CIN2/CIN3 among 95 women with ASC-US (8.4%), and 23 cases of CIN2/CIN3 among 104 women with LSIL (22.1%). In the group of women with ASC-US, the sensitivity and specificity in diagnosing CIN2/CIN3 were 87.5% and 79.5% for the HPV test and 62.5% and 93.1% for p16/Ki-67. Among women with LSIL, the sensitivity and specificity in the diagnosis of CIN2/CIN3 were 87% and 34.7% for the HPV test and 69.6% and 75.3% for immunocytochemistry. Superior performance was observed for p16/Ki-67 double staining, especially among women under 30 for whom the test had an area under the ROC curve of 0.762 (p<0.001). Both p16/Ki-67 double staining and the hr-HPV DNA test had similar performance in predicting high-grade cervical intraepithelial neoplasia among women with ASC-US. The best performance was observed in women aged >30 years. In younger women (≤30 years) with LSIL, p16/Ki-67 had greater accuracy in identifying precursor lesions. Among women >30 years diagnosed with LSIL, the two methods showed similar performance. Among malignant neoplasias, cervical cancer has the greatest potential for prevention. However, it currently remains an important public health problem, especially in developing countries where it is associated with high incidence rates and mortality. Worldwide, it is the third most common cancer among women. The Pap smear has helped reduce the incidence of cervical cancer, especially in developed countries where organized screening programs were implemented , but theDifferent population-based studies assessing the role of different molecular assays for high-risk HPV (hr-HPV) detection in primary screening based on a cervical cancer prevention proposal have provided a significant increase in sensitivity and reproducibility compared to the Pap test . Despitethe Cobas 4800 test  which was approved by the FDA in 2014 for use in primary screening. This system allows the detection of 14 hr-HPV genotypes by polymerase chain reaction (PCR). In addition, it discriminates genotypes 16 and 18, which are associated with 70% of invasive cervical carcinomas [Hybrid Capture 2 test (hc2) test  with the Cobas 4800 test have shown no significant differences in the accuracy of the two methods [Recently, new tests have been introduced for the molecular detection of HPV DNA, including  methods –13.CINtec PLUS to identify positivity rates of 26.8% in normal histological specimens and 46.5%, 82.8% and 92.8% in patients with CIN1, CIN2 and CIN3 biopsies, respectively [CINtec PLUS and Cobas 4800 test) in the diagnosis of CIN2/CIN3 among women with ASC-US cytology and LSIL.Even within the limitations of the Pap test, the literature indicates that the presence of CIN2+ may occur in 5 to 22% of patients with a cytological diagnosis of atypical squamous cells of undetermined significance (ASC-US) , 14, 15 ectively . HoweverCINtec PLUS) and the molecular test for hr-HPV DNA (Cobas 4800 test) for the identification of CIN2/CIN3 lesions in women with a cervical cytology of ASC-US or LSIL.The objective of the present study was to compare the accuracy of p16/Ki-67 double staining . The results were categorized as ASC-US or LSIL, according to the criteria established in 2001 by the Bethesda System [In this prospective, cross-sectional study, women were examined between February 2012 and July 2013 at the Departments of Pathology and Prevention of The Barretos Cancer Hospital (HCB), Barretos, São Paulo, Brazil. The study included women over 18 years of age who underwent the Pap test for cervical cancer screening using liquid-based cytology a System .All of the women received written explanations and were verbally informed about the study purpose and procedures. Written consent was obtained previously and this study was approved by the Ethics Committee in Research of The Barretos Cancer Hospital. All of the identifying information for the patients was maintained confidentially and encrypted in a database to ensure data confidentiality and privacy of the participants.International Federation for Cervical Pathology and Colposcopy—Rio de Janeiro, 2011 [Women with LSIL and ASC-US were referred for colposcopy at the gynecology clinic of the Department of Prevention of HCB. The colposcopy results were classified according to the nomenclature of the Prepstain BD for the immunocytochemistry analysis (ICQ) using the CINtec PLUS Cytology kit  according to the manufacturer's instructions. All slides were examined by two different observers (JCP-R and CS-N). The results of discordant cases were determined by a consensus of the two examiners. Samples with one or more cervical epithelial cells that simultaneously showed brownish cytoplasmic immunostaining (p16) and red nuclear immunostaining (Ki-67) were classified as positive regardless the of morphological appearance of the cells  according to the manufacturer's instructions. The Cobas HPV test combines the polymerase chain reaction (PCR) in real time and nucleic acid hybridization in a single analysis to detect 14 hr-HPV DNA: HPV-16 and HPV-18 individually and the other 12 types pooled .The residual cells of the cell pellet prepared for the liquid-based slide (SurePath) were used for the hr-HPV DNA analysis with the p less than 0.05 was considered statistically significant.The chi-square test was used to evaluate the association between HPV DNA testing and immunocytochemical results with colposcopy and biopsy. Confidence intervals (95%) of the indicators of accuracy were calculated, and the performance of the diagnostic tests for predicting the presence of high-grade cervical intraepithelial neoplasia (CIN2/CIN3) was evaluated by the Receiver Operating Characteristic (ROC). Areas under the ROC curve (AUC) were compared using the chi-square test and dividing the study population according to the result of the initial Pap test and adjusting for the age of the women. A value of A total of 201 women (96 with ASC-US and 105 with LSIL cytology) aged between 18 and 76 years (mean = 39.2) were eligible for inclusion in the study and underwent colposcopy, HPV testing and immunocytochemistry. The time between sample collection for cervical cytology and colposcopy ranged from 23 to 80 days . Tissue samples were obtained from cervical biopsy and/or EC in 151 women (75.1%). Cases with a satisfactory colposcopy without visible lesions were considered normal. Two samples  were excluded from the analysis due to inconclusive results on the histologic examination. There were 8 cases of CIN2/CIN3 among 95 women with ASC-US cytology, and 23 cases of CIN2/CIN3 among 104 women with LSIL . No caseValid HPV test results were obtained for 191 samples, with an overall positivity rate of 49.2%. Positive HPV tests were obtained in 27.2% and 69.7% of the women with ASC-US and LSIL cytology, respectively. The distribution of HPV types among patients without epithelial lesions and CIN1 or CIN2/CIN3 are depicted in Approximately 25% of the cases (23.9% in 201 samples) stained positive for p16/Ki-67 (cytoplasmic/nuclear), and women with ASC-US and LSIL cytology had positive p16/Ki-67 indices of 11.5% and 35.2%, respectively. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the ROC curve for both HPV testing as immunocytochemistry are shown in The goal of this study was to evaluate the performance of two tests in predicting the presence of clinically relevant lesions among women with Pap test results indicative of mild atypia. The results of this study were intriguing because they revealed the importance of incorporating well-tested, new technologies in areas with a high prevalence of cervical lesions. These technologies can complement or even replace the use of cervical cytology in screening for precursor lesions and cervical cancer due to the previously mentioned limitations of the Pap test and ratified by different revisions that showed huge variations in rates of sensitivity and specificity , 3.The cytology classified as ASC-US remains a challenge from the standpoint of clinical management by presenting imprecise diagnostic criteria with questionable levels of agreement among experienced pathologists  and ofteThe accuracy of the Cobas 4800 test to predict the presence of CIN2/CIN3 among women with ASC-US cytology was consistent with previous studies –11. AddiCompared with the hr-HPV DNA test, the p16/Ki-67 immunocytochemical reaction provided better specificity but with a decreased rate of sensitivity. Although hr-HPV DNA testing can be used to identify a greater number of patients with significant cervical lesions, a larger contingent of women are unnecessarily referred for colposcopy compared with the use of immunocytochemical analyses. Some studies have demonstrated variables indices of sensitivity (ranging from 64% to 98%) and specificity (ranging from 43% to 81%) for the immunocytochemical test , 18, 22.This low sensitivity may be attributed to the conservative parameters to ascertain p16/Ki-67 positive reactions. The cases in which we had difficulty in discriminating a weak cytoplasmic p16 staining from a background reaction, we classified the reaction as negative. Another important point to be addressed is the difficulty in classifying the reaction in three-dimensional cellular groups or metaplastic cells.To predict the presence of CIN2/CIN3 among women with ASC-US cytology, the two methods produced similar results, especially in women over 30 years old. Although the analyses used to identify hr-HPV DNA (Cobas 4800) demonstrated a greater area under the ROC curve, both tests provided a value of 0.919 in the overall analysis and among women >30 years old, and there was no statistically significant difference with respect to the accuracy of the immunocytochemistry.The presence of precursor lesions (CIN2/CIN3) in women with a Pap test categorized as LSIL is not negligible, with rates ranging from 9% to 30% –26. We fCobas 4800 test for predicting CIN2/CIN3 was 87% and 34.7%, respectively. Compared to women with ASC-US cytology, hr-HPV DNA test did not show the same performance in terms of specificity probably because of the high rate of viral infection by high-risk subtypes in the latter group. The use of the test to detect hr-HPV DNA among women with LSIL is not necessarily the most appropriate alternative to discriminate which women are at increased risk for clinically relevant cervical lesions. The hr-HPV DNA test must be used judiciously because its associated significant increase in the number of women referred to colposcopy and unnecessary procedures could negatively impact public health costs, and there is also the potential psychological distress that the diagnosis of sexually transmitted infection can generate among women not adequately informed about the natural history of HPV.Among women with LSIL cytology, the sensitivity and specificity of the The performance of the CINtec PLUS test in predicting the presence of high-grade squamous intraepithelial lesions among women with LSIL cytology demonstrated a sensitivity and specificity of 69.6% and 75.3%, respectively. The test (p16/Ki-67) maintained the same levels of sensitivity (85.7%) compared with HPV testing; women aged ≤30 years had a comparatively much higher specificity of 66.7%. Additionally, individualizing the accuracy of the CINtec PLUS test to identify CIN3, among women with LSIL cytology, the test performance demonstrated 100% sensitivity and 69.4% specificity; NPV was 100% regardless of age, indicating improved performance of the test with the severity of the lesion.Schmidt et al. achieved significant results analyzing the accuracy of p16/Ki-67 dual staining in predicting the diagnosis of CIN3 between LSIL cytology with sensitivity and specificity values of 95.8% and 68%, respectively . ConsistIn summary, both of the analyzed methods (hr-HPV Cobas 4800 test and CINtec PLUS) showed similar performance in the prediction of high-grade cervical intraepithelial neoplasia among women with cytology diagnosed as ASC-US, with the best performance obtained among women aged >30 years. Among younger women (≤30 years) with LSIL, p16/Ki-67 dual staining had greater accuracy in identifying precursor lesions, especially CIN3 as compared to molecular testing for high-risk oncogenic HPV. Among women older than 30 years and diagnosed with LSIL, the methods showed similar performance.S1 File(XLSX)Click here for additional data file.


Background. HIV-infected adolescents may be at higher risk for high-grade cervical lesions than HIV-uninfected adolescents. The purpose of this study was to compare the prevalence of high-risk HPV (HR-HPV) infections and Pap smear abnormalities between these two groups. Methods.In this cross-sectional study, we compared the HPV DNA and Pap smear results between 35 HIV-infected and 50 HIV-uninfected adolescents in order to determine the prevalence of HR-HPV genotypes and cervical cytological abnormalities. Comparisons were made using Pearson χ2 and independent-samples t-tests analyses, and associations between demographic and behavioral characteristics and HPV infections were examined. Results. HIV-infected participants were more likely to be infected with any HPV  and with at least one HR-HPV , and to have multiple concurrent HPV infections . HPV 16 and 18 were relatively underrepresented among HR-HPV infections. Abnormal Pap test results were more common among HIV-infected participants . A history of smoking was associated with HR-HPV infection. Conclusions. HIV-infected adolescents have an increased risk of infection with HR-HPV and of Pap test abnormalities. The majority of HR-HPV infections among our participants would not be prevented by the currently available vaccinations against HPV. Human papillomaviruses (HPV) cause cervical cancer . The gloWomen who are infected with HIV are at increased risk of dying from cervical cancer . They arScreening guidelines also differ between adults and adolescents. Due to the high rate of spontaneous clearance of low-grade cervical lesions and the risks associated with over-treatment among adolescents, it is recommended that screening does not begin until the age of 21 , 12. HIVWe compared the HPV DNA and Pap smear results between 50 HIV-uninfected and 35 HIV-infected adolescent females from Masiphumelele, South Africa, in order to determine the prevalence of HR-HPV, multiple concurrent HPV infections, and cervical cytological abnormalities. We examined associations between these results and potential risk factors including sexual activity, contraception use, and smoking.M = 19.06 years; S.D. = 1.48; IQR = 18.00–20.00). While definitions of the age range that constitutes adolescence vary, we defined it in accordance with the American Academy of Pediatrics that categorizes late adolescence to extend to the age of 21 [Between October 2012 and February 2014 we conducted a cross-sectional prevalence study in which cervical specimens for Pap smear and self-collected vaginal swabs for HPV DNA testing were collected from 50 HIV-uninfected and 35 HIV-infected sexually active South African adolescent females aged 17–21 (ge of 21 . ExclusiAll participants signed informed consent (aged 18 and older) or signed adolescent assent documents (aged 17) to accompany parental consent forms in order to participate in the study. This study was approved by the Research Subjects Review Board at the University of Rochester and the Human Research Ethics Committee at the University of Cape Town.For self-sampling, patients were instructed to insert a Dacron swab high into the vagina and twirl it for 10 seconds. Self-sampling was conducted in private. Samples were stored in Digene transport medium and DNA was extracted using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche Diagnostics). HPV genotyping was conducted using Roche's Linear Array HPV Test. This kit detects 37 HPV genotypes  includin Trichomonas) were referred for appropriate treatment. Most recent CD4 test results were obtained from the local government clinic where HIV-infected participants receive care.Pap smear results were reported in accordance with the Bethesda system . All posχ2 and independent-samples t-tests analyses, and associations between demographic/behavioral characteristics and HPV infections were examined using Spearman correlations. All analyses were performed using IBM SPSS 22.0.HIV-infected and HIV-uninfected youth were compared using Pearson We found a large burden of HPV infection in our study cohort. Overall 64.7% of participants had at least one HPV infection and 38.8% had at least one HR-HPV infection. Multiple concurrent HPV infections were common with 41.2% of all participants testing positive for two or more HPV genotypes (63.6% of HPV-infected participants were infected with greater than one genotype). Multiple concurrent HR-HPV infections were found in 24.7% of all participants and 63.6% of HR-HPV-positive participants were infected with two or more HR-HPV genotypes. Eleven out of the 85 participants (12.9%) had five or more concurrent HPV infections detected. Likewise, 24.2% of HR-HPV positive participants had three or more concurrent HR-HPV infections.Non-vaccine HR-HPV genotypes (those other than 16 and 18) were notably common among our cohort. Among HR-HPV positive participants, only 12.1% were infected with exclusively vaccine genotypes, while 51.5% were infected with exclusively non-vaccine genotypes (36.4% were infected with a combination of vaccine and non-vaccine genotypes).P = 0.049), among all participants with one or more HR-HPV infections, HIV-infected individuals were more likely to be infected with a non-vaccine HR-HPV genotype (P = 0.006). Numerous significant differences in HPV infection were identified between the HIV-infected and HIV-uninfected groups. HIV-infected participants were more likely to be infected with any HPV, to be infected with at least one HR-HPV, and to have multiple concurrent HPV infections. Although HPV 16 was more prevalent in the HIV-infected group .Overall, 18.8% of our cohort had abnormal Pap tests. Pap smear abnormalities were found to be more common among HIV-infected participants than their HIV-uninfected counterparts. No study participants were found to have high-grade squamous intraepithelial lesions (HSIL). There were two HIV-infected youths observed with atypical squamous cells—cannot rule out HSIL (ASC-H) and six with low-grade squamous intraepithelial lesions (LSIL) compared to zero and two, respectively, among HIV-uninfected youth. 3 . The average CD4 for those on ARVs was 301 and for those not on ARVs was 557 (P = 0.012). We did not identify any significant relationship between ARVs and HPV infection. Likewise, CD4 count was not found to be significantly associated with HR-HPV infection or abnormal Pap test.Among the HIV-infected participants, 22.9% were receiving antiretroviral medications (ARVs) at the time of testing. The average CD4 count among all HIV-infected participants was 490/mmP = 0.049). A history of smoking was positively associated with HR-HPV infection (P = 0.024). Methods of contraception used, number of lifetime sexual partners, and number of sexual partners reported in the six months prior to enrollment were not significantly associated with HPV infection.Among our bivariate analyses, we found the older members of our cohort to have a greater number of multiple concurrent HR-HPV infections (P = 0.001). Marais et al. have reported similar differences in HR-HPV prevalence between HIV-infected and HIV-uninfected South African women from an older cohort (mean age: 35) [Our results show an increased risk of infection with HR-HPV and associated Pap smear abnormalities among HIV-infected adolescents compared to their HIV-uninfected counterparts. This is consistent with previous data from South Africa. A recent report comparing the distribution of HPV genotypes between HIV-infected and HIV-uninfected South African women aged 17–65 years found differences in HR-HPV prevalence across all age groups including 17–19-year-olds who were found to have an HR-HPV prevalence of 75% and 60%, respectively [age: 35) . The lonAlthough identifying the HR-HPV genotypes associated with cases of invasive cervical cancer provides the most critical information regarding the proportion of cases preventable by vaccination, the high relative prevalence of non-vaccine HR-HPV compared to the vaccine genotypes in our cohort does demonstrate that the currently available vaccines against HPV would not prevent the majority of HR-HPV infections among our study population. Still, without longitudinal data demonstrating incident and/or persistent cervical disease associated with non-vaccine HR-HPV infection, the significance of this observation is uncertain.HIV-infected women in sub-Saharan Africa are among the groups at highest risk of developing cervical cancer. Although adolescents are generally at low risk for cervical cancer , those wAll cervical cancer screening strategies endeavor to identify cervical dysplasia prior to it reaching an invasive stage while also protecting women from the risks of overtesting and overtreatment. Tremendous efforts have been made to identify cost-effective approaches to the follow-up of negative Pap tests and low-grade Pap smear abnormalities, resulting in recommendations that utilize HPV DNA testing to extend intervals between Pap tests , 12. TheIn this study, self-collected vaginal swabs were used to provide samples for HPV DNA testing. Agreement between the results from clinician-collected and self-collected specimens has been reported to be high in several studies , even amOur study setting has very high density of HIV and HPV infection, providing a unique opportunity to investigate cervical cancer risk among HIV-infected adolescents. Still, our study has several limitations. Although numerous significant data were found, our sample size is relatively small and may be insufficient to identify real differences between study groups. Our current sample size limits the types of multivariate analyses that could be performed to accompany the descriptive statistics and bivariate correlations presented. Subsequent research should seek to examine differences across HIV status while accounting for demographic and behavioral variables. Only 22.9% of our HIV-infected participants were on ARVs and our cohort had relatively high CD4 counts so we are unable to correlate our findings with levels of immunosuppression. Previous research has shown that more advanced HIV disease is strongly associated with increased cumulative HPV prevalence, more advanced cervical dysplasia, and an increased rate of progression of cervical disease , 28–30. Our results underscore the overall high-risk for HPV-related cervical disease in our study population and the significant potential difference in risk for cervical cancer between HIV-infected and HIV-uninfected young women in South Africa. The majority of HR-HPV infections among our study participants would not be prevented by the currently available vaccinations against HPV although the impact of these infections on incident advanced cervical disease is not clear. An optimal, evidence-based approach to cervical cancer screening for HIV-infected adolescents may include the use of HPV testing which has the potential to allow for an increase in the interval between Pap tests. This would have particular benefits in low-resource settings such as Masiphumelele. Longitudinal studies with this and similar cohorts are required to quantify the difference in risk for high-grade cervical lesions between HIV-infected and HIV-uninfected adolescents and to determine if HPV testing can be effectively integrated into a cervical cancer-screening program for adolescents with HIV.


To assess the utility of makorin ring finger protein 1 (MKRN1) as a marker of cervical pathology.INK4a. Results of liquid-based cervical cytology, immunostained cell block sections, and human papillomavirus (HPV) hybrid capture  were analyzed. Clinical outcomes were analyzed overall and in subsets of specimens yielding atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions.A PROspective specimen collection and retrospective Blinded Evaluation study was conducted. Liquid-based cytology samples were collected from 187 women, embedding all residuals as cell blocks for immunohistochemical staining of MKRN1 and P16 Makorin ring finger protein 1 positivity and grades (1–3) of cervical intraepithelial neoplasia (CIN) increased in tandem , reaching 92.3% in invasive cancer. Sensitivity, specificity, positive predictive value, and negative predictive value in detecting CIN2+ via MKRN1 were 73.8%, 76.8%, 75.6%, and 75.0%, respectively. The performance of liquid-based cytology was poorer by comparison , and HPV assay  displayed lower specificity (67.7%). Combined HPV + MKRN1 testing proved highest in sensitivity, specificity, positive predictive value, and negative predictive value , whereas corresponding values for cytology + HPV  and cytology + MKRN1  were all similar. In instances of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions, the HPV + MKRN1 combination performed best by above measures , followed by cytology + MKRN1 .Makorin ring finger protein 1 displayed greater sensitivity and specificity than liquid-based cytology and proved more specific than HPV assay. In combination testing, MKRN1 + HPV showed the highest sensitivity and specificity levels. The MKRN1 biomarker may be a useful adjunct in primary cervical cytology screening. Unfortunately, HPV screening has low specificity in this setting. Although such infections are common and are apt to resolve naturally within 1 to 2 years, both transient bouts and persistent infections (accounting for high-grade CIN)4 yield positive test results.Given that human papilloma virus (HPV) infection is implicated in cervical carcinogenesis, a recent study has found HPV deoxyribonucleic acid (DNA) testing more sensitive than cervical cytology, detecting high-grade CIN earlierINK4a (a cell-cycle regulatory protein), both of which have been identified in prior studies as markers of CIN.8 Conversely, p16INK4a immunoreactivity is also observed in endocervical or metaplastic cells and in benign atrophic cells, requiring attention to cell morphology when interpreting stained specimens.9Many researchers are now focused on developing more effective screening tests for CIN detection, hoping to improve the specificity of cytologic preparations and HPV tests while maintaining the generally high respective sensitivities. Biomarkers of HPV-related genes strongly expressed in carcinogenesis are of particular interest. Prime examples include Ki-67 (involved in cellular proliferation) and p1610 Makorin ring finger protein 1 also blocks cancer cell death by inducing ubiquitination and thus promoting degradation of Fas-associated protein with death domain, a key element in death receptor-activated extrinsic apoptosis. Because MKRN1 and Fas-associated protein with death domain participate in necrosome formation and necroptosis regulation, downregulation of MKRN1 understandably has been shown to have a major inhibitory effect on tumor enlargement in breast cancer. Furthermore, MKRN1 messenger ribonucleic acid levels are significantly higher in cancerous than in normal cervical tissue.11Makorin ring finger protein 1 (MKRN1) is a transcriptional coregulator, an E3 ligase, and a negative regulator of tumor suppressor genes p53 and p21. It has been noted that a reduction in MKRN1 induces growth arrest by activating p53 and p21.INK4a immunostaining) were evaluated in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy in diagnosing CIN2+. For this purpose, residual liquid-based cytology samples enabled immunostaining of MKRN1 and p16INK4a biomarkers.The current study was conducted to determine if MKRN1 immunohistochemical (IHC) staining is a viable adjunct in diagnosing cervical cancer or its precursor lesions. Specifically, 4 diagnostic procedures . Exclusions were as follows: age <18 years, prior hysterectomy, previous cancer of noncervical origin, treatment for CIN or invasive cancer within last 5 years, chronic illness with immunocompromise, or refusal to consent/participate. The clinical performance of each test method was determined retrospectively, based on histologic findings in punch biopsy or hysterectomy specimens.Specimens were prospectively collected between July, 2013 and February, 2014, following approval by the Institutional Review Board for Clinical Research at Gangnam Severance Hospital. The study population (n = 189) consisted of women ≥18 years old who were referred to the above facility for abnormal cervical cytology results; who were admitted with benign conditions  for hysterectomies; or who visited the hospital for regular checkups and routine cervical cytology screening. Each enrolee was subjected to cervical cytology screening, HPV assay, and immunostaining for MKRN1 and p16A liquid-based cervical cytology sample was obtained from each patient via Cytobrush device . Two pathology specialists (same institution affiliates) rendered all diagnoses, based on the Bethesda system (2001). In the event of conflicting results , higher grade lesions prevailed for analysis.The Hybrid Capture 2 (HC2) assay , which is designed to detect 13 types of high-risk HPV, was routinely performed. Results were expressed as a ratio between light emitted from a test specimen and from 1 pg/mL HPV DNA (average of 3 control specimens). For example, 1 relative light unit would correspond with 1 pg/mL HPV DNA in a specimen tested. Because HC2 assays cannot distinguish between single and multiple infections, nor distinguish between HPV types 16 and 18, the cobas 4800 HPV test  was also performed. Results were reported as negative, type 16 or 18, and other types. Positive HPV testing was equated with HPV positivity by HC2 assay or detection of any HPV type (HPV 16 or 18 and others).Punch biopsies were obtained by a specialist in colposcopy to confirm normal cytology results or corroborate the presence of high-risk HPV infection. Patients with CIN1 or lower grades were followed without further treatment. In every patient with CIN3+ and in most with CIN2, either a loop electrosurgical excision procedure or a hysterectomy was done. Those diagnosed with invasive cervical cancer underwent radical hysterectomy. If punch biopsy and loop electrosurgical excision procedure or hysterectomy specimens resulted in conflicting diagnoses, the highest grade prevailed for analysis.INK4a were prepared from residual samples , based on a method described in a number of previous studies. Specifically, residual PreservCyt Solution (Cytyc) was centrifuged , discarding the supernatant and resuspending the pellet in 5 to 8 drops of plasma. Next, 2% calcium chloride was added, mixed well, and left to coagulate (20 minutes). The resultant clot was wrapped in lens paper, placed in a tissue cassette, and fixed (10 minutes) in 10% neutral buffered formalin. Completed preparations were sectioned at 4.0 μm and stained with hematoxylin and eosin. Cellular morphology and degree of preservation were assessed.Cell blocks for immunostaining of MKRN1 and p16To determine MKRN1 expression, prepared cell blocks were again sectioned at 400 μm, and IHC staining was performed using rabbit anti-MKRN1 antibody and affinity purified makorin-1 antibody . The UltraVision LP Detection System kit  was used for indirect staining, using a biotin-linked secondary antibody labelled with horseradish peroxidase. The peroxidase reacts with 3,3′-diaminobenzidine tetrahydrochloride, catalyzing a color change, and the slides are counterstained with hematoxylin. Sections were interpreted by a pathology specialist, equating a positive result with visible nuclear staining  and CINtec p16-INK4a Cytology kit . The slides were then incubated (30 minutes) with secondary goat anti-mouse antibody (Dako), followed by 3,3′-diaminobenzidine tetrahydrochloride (Dako) incubation (10 minutes) and finally counterstaining with Mayer hematoxylin.Expression of p16INK4a) using standard software . Based on calculated sensitivity and specificity values, diagnostic accuracy of each method was determined from receiver operating characteristic curves. Identical analyses were performed for each 2-method combination tested in the entire cohort and in patient subsets showing atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous intraepithelial lesions (LSIL). Each result was expressed as mean, with a 95% confidence interval. Statistical significance was set at P < 0.05.In diagnosing CIN2+, sensitivity, specificity, PPV, and NPV were calculated for each test method  and 12 (21.4%), respectively. Of 38 patients with CIN1, 30 showed positivity by cytology, HPV assay, and p16INK4a immunostaining, whereas 12 tested positive for MKRN1. Of 32 patients (17.1%) diagnosed with CIN2, 29 (90.6%), were positive by cytology and HPV assay, with similar rates for MKRN1 and p16INK4a . In 47 patients (25.1%) with CIN3/CIS, positive results were recorded as follows: cytology, 44 (93.6%); HPV assay, 46 (97.9%); MKRN1, 35 (74.5%); and p16INK4a, 36 (76.6%). In 14 patients (7.5%) with invasive cancer, positivity totals for cytology, HPV test, and MKRN1 marker were the same at 13 (92.9%), with 12 (85.7%) positive results for p16INK4a.Performance levels of the 4 cervical cancer screening tests under investigation are shown by histologic diagnoses in Table INK4a proved similar in sensitivity (74.2% versus 73.1%) but lower in specificity (53.2% versus 74.5%). Used in combination, cytology and HPV assay (cytology + HPV) showed the highest sensitivity (90.3%) but the lowest specificity (66.0%) of all combinations studied. Combined use of HPV assay and MKRN1 immunostaining (HPV + MKRN1) showed the highest specificity (87.2%). Equivalent sensitivity (71.0%) and specificity (77.7%), however, were observed for cytology and MKRN1 marker (cytology + MKRN1) or HPV assay and p16INK4a marker (HPV + p16INK4a) in combination. The second lowest specificity (69.1%) was encountered with cytology and p16INK4a marker (cytology + p16INK4a) in combination, with cytology + HPV showing the lowest specificity. Combined marker screening (MKRN1 + p16INK4a) had the second highest specificity (80.9%) but the lowest sensitivity (62.4%). The 2-method combination with the highest specificity was HPV + MKRN1. In terms of diagnostic accuracy, HPV assay and MKRN1 immunostaining were the most accurate methods in single use , whereas 2-method accuracy was highest (78.1%) for cytology + HPV and HPV + MKRN1.The goal of this study was to accurately diagnose precancerous cervical pathology. To determine the diagnostic accuracy of the 4 screening tests, we calculated sensitivity, specificity, PPV, NPV, and diagnostic accuracy for both individual tests and 2-method combinations Table . Based oINK4a markers were similar , but MKRN1 showed the highest specificity (62.5%). In combination, cytology + HPV earned the highest sensitivity (100%) but the lowest specificity (41.7%), whereas HPV + MKRN1 showed high specificity at 81.3%. As individual tests, HPV assay and MKRN1 immunostaining were the most accurate , as with patients overall, whereas cytology was least accurate (39.7%). Of the 2-method combinations analyzed, HPV + MKRN1 showed the highest diagnostic accuracy (73.1%), surpassing that of cytology + HPV (64.1%).In patients with ASCUS or LSIL patients, cytology and HPV assay again showed the highest individual sensitivity (100%), with low specificity. Sensitivities of MKRN1 and p16INK4a as a novel marker for cervical neoplasia. In a large-scale randomized trial, targeting of p16INK4a reportedly increased diagnostic specificity and correlated strongly with histologic results.7 Assessment of p16INK4a overexpression (via immunostaining) and HPV assay in combination yielded better diagnostic accuracy than conventional cervical cytology and HPV testing. Consequently, the pursuit of useful adjuncts to existing methods is still a high priority in ongoing research. In a study conducted by Lee et al11, overexpression of the MKRN1 protein correlated with cervical cancer in vivo. Another previous study has also shown that MKRN1 expression levels in cervical tissue are proportional to tumor stage or grade of tumor, correlating positively with pAKT and negatively with phosphatase and tensin homolog (PTEN) expression levels.12To compensate for the low specificities of cytology and HPV testing, researchers have focused on p16Outcomes of the current study indicate that HPV and MKRN1 determinations are similar in terms of diagnostic accuracy (∼74%), each surpassing cervical cytology (67.4%). In dual applications (cytology + HPV and HPV + MKRN1), the diagnostic accuracies, however, were comparable (78.1%) while specificities differed. The combination of cytology + HPV had the lowest specificity, but individual sensitivities were highest . Thus, the MKRN1 marker essentially complements the low-specificity, high-sensitivity nature of HPV assay, raising overall specificity from 54.3% to 87.2% and delivering the highest diagnostic accuracy. Hence, the benefit conferred justifies the effort and cost of an added test.7; therefore, unnecessary procedures may be avoided by conservative measures . Nonetheless, it is alarming that a high percentage of patients  in our cohort with these cytologic abnormalities eventually were diagnosed as CIN1+, and CIN2+ was confirmed histologically in 30 (39.0%) of these patients. This suggests a clear propensity for later development of invasive cancer, making it imperative to separate those who require active intervention from others for whom observation is sufficient. As such, we investigated the clinical performance of screening tests in patient subsets showing ASCUS or LSIL on cytology. Outcomes were similar to those observed in patients overall. In combined HPV + MKRN1 testing, specificity was nearly 2-fold that of cytology + HPV (81.3% versus 41.7%), resulting in the highest diagnostic accuracy (76.5%).The most difficult scenarios in cervical cancer screening are those complicated by ASCUS and LSIL. Previous studies contend that such changes will likely disappear13 Because younger women typically have plans of future childbirth, the increased likelihood of procedure-related obstetric morbidity, such as preterm birth or incompetent internal os, may become important.14 Moreover, cytology results indicative of ASCUS, LSIL, or HPV infection are a potential source of psychologic stress in women. Therefore, a physician's decision to opt for follow-up observation (with patient reassurance) or halt progression to invasive cancer through aggressive testing and treatment is critical. The exceptional specificity of HPV + MKRN1 in patients of questionable status (ASCUS or LSIL) is particularly noteworthy.According to epidemiologic studies of HPV, infection rates are higher in sexually active women <35 years of age, although most episodes resolve naturally.15 As the proportion of patients with invasive cancer or rapidly progressive high-grade CIN (transitioning to invasive cancer) declines, owing to effective curtailment of HPV 16 and 18 serotypes, a commensurate decline in the PPV of cytology is expected. Thus, the preferred test for primary cervical cancer screening is gradually shifting from cytology to the HPV DNA assay, despite the low specificity. Makorin ring finger protein 1 may then play a key adjunctive role in cervical screening, as demonstrated herein.Human papillomavirus vaccination coverage has increased significantly after approximately 8 years of usage for cervical cancer. The US Food and Drug Administration granted approval of Gardasil in 2006 and Cervarix in 2009.16 to determine the accuracy of the Pap tests showed wide variations in sensitivity (11%–99%) and specificity (14%–97%). In contrast, HPV assay is an objective and highly sensitive automated procedure, with limited opportunity for medicolegal claims because of disputed results.Cytology screening relies on subjective assessments that prove disproportionately unsatisfactory in less skilled hands. Trained professionals must carefully search for atypical cells, which may be obscured by a dearth of normal epithelial elements. Moreover, intra- or interobserver discrepancies are possible in interpreting ASCUS and high-grade squamous intraepithelial lesion. A systematic review of 62 studies conducted by Fahey et alThe current study is the first to report the features and benefits of MKRN1 as a diagnostic marker for invasive cervical cancer/CIN. This study was free of selection bias, given prospective collection of specimens and treatment guidelines developed after the study was planned. All data were analyzed retrospectively, based on histology reports. Makorin ring finger protein 1 IHC staining was found to be an effective test method that complemented the low specificity of cytology and HPV assay. Residual specimens from liquid-based cytology or cobas HPV analysis may be used for staining, without the added effort, discomfort, or anxiety during gynecologic examinations. Of particular importance, this method may curb unnecessary colposcopically directed biopsy and referral rates, thereby reducing medical costs while safely extending screening intervals.In terms of future study considerations, the relationship between clinical performance of HPV + MKRN1 and patient age should be investigated, assessing study population subsets. In addition, polymerase chain reaction-based experiments are needed to confirm the possibility of more convenient method. Above all, cost-effective analysis is required to clarify the best screening strategy including MKRN1 IHC method for diagnosing CIN2+. Follow-up analysis of whether MKRN1 presence or absence impacts progression to high-grade CIN is another important issue.In conclusion, comparative clinical performances of MKRN1 IHC staining and currently available cervical cancer screening tests indicate that HPV + MKRN1 is the ideal test combination, despite equivalent accuracy of cytology + HPV. The MKRN1 and HPV methods complement 1 another, considering the high sensitivity of HPV assay and the high specificity of the MKRN1 biomarker. Furthermore, the HPV + MKRN1 combination was most accurate for diagnosing CIN2+ in ASCUS/LSIL patient subsets. We anticipate that MKRN1 IHC staining will ultimately emerge as a useful adjunct to routine cervical cancer screening.


INK4a expression is used as a surrogate marker for HPV infection, there is controversy as to whether p16INK4a reliably indicates HPV infection. Here, to evaluate the accuracy of p16INK4a expression for determining HPV infection and the prognostic value of HPV infection and p16INK4a expression for HNSCC survival, especially oropharyngeal squamous cell carcinoma (OPSCC) survival, 150 fresh-frozen HNSCC samples were analyzed for HPV DNA, E6/E7 mRNA and p16INK4a expression by polymerase chain reaction and immunohistochemistry. p16INK4a expression was scored from 0 to 4 according to the percentage of p16INK4a-positive cells, with overexpression defined as >40% positive cells. Of the 150 tumor samples tested, 10 tumors were nasopharyngeal, 53 oropharyngeal, 39 hypopharyngeal, 24 laryngeal and 24 were located in the oral cavity. HPV DNA was detected in 47 (31.3%) samples, but only 21 also exhibited HPV mRNA expression. Inter-rater agreement was low between p16INK4a expression and HPV DNA presence and between p16INK4a expression and HPV mRNA expression, but was good between the combination of HPV DNA status and p16INK4a overexpression and HPV mRNA expression. Three-year recurrence-free survival was significantly higher for OPSCC patients who were HPV DNA-positive than for OPSCC patients who were HPV DNA-negative (P=0.008) and for OPSCC patients over-expressing p16INK4a than for without overexpressing p16INK4a (P=0.034). Multivariate analysis revealed that T1-3 stage and the combination of HPV DNA positivity and p16INK4a overexpression predicted significantly better recurrence-free survival. This combination is a more accurate marker for active HPV infection in HNSCC than HPV DNA status or general p16INK4a-positive status alone and offers a useful and reliable method for detecting and determining the prognosis of HPV-related HNSCC.Head and neck squamous cell carcinoma (HNSCC) patients with human papillomavirus (HPV) infection have better prognosis than those without HPV infection. Although p16 Each year, 600,000 new cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed worldwide . Common E6/E7 mRNA transcripts is regarded as the gold standard for the presence of clinically relevant (active) HPV  HPV , the reqINK4a has been proposed as a surrogate marker for HPV infection: its over-expression is thought to reflect the presence of biologically active HPV infection given that functional inactivation of pRb by viral E7 induces p16INK4a upregulation. Detection of p16INK4a expression can also be performed using formalin-fixed, paraffin-embedded (FFPE) samples , sporadic (<5% of cells positive), focal (<25% of cells positive) or diffuse (>25% of cells positive) (INK4a expression in tumors as strong and diffuse when ≥70% of cells (cytoplasm and nuclei) are stained  . Other s stained –17, whilpositive . These dgulation . Bussu eINK4a expression and the value of both HPV-positive status and p16INK4a expression levels for HNSCC prognosis using tissue samples from a well-characterized cohort of Japanese patients with HNSCC receiving curative treatment. We measured the presence of HPV DNA, HPV E6/E7 mRNA expression using fresh-frozen samples and measured p16INK4a expression using FFPE samples.In this study, we evaluated the relationship between HPV infection and p16INK4a in tumor tissue. Based on these criteria, 150 patients treated by the Department of Otorhinolaryngology, Head and Neck Surgery, University of the Ryukyus, Japan were recruited between October 2006 and June 2013. In contrast to our previous study on HPV status and squamous cell carcinoma antigen (SCCA), this study involved a greater total number of cases who met the inclusion criteria and we also updated the prognostic information of some of the patients reported previously ; receiving curative treatment of surgery alone, surgery combined with radiation therapy (RT) or chemoradiotherapy (CRT), concurrent chemoradiotherapy (CCRT) or RT alone with >66 Gy of total dosage; and complete remission after primary treatment. The treatment modalities were determined according to tumor location, tumor stage, response to induction chemotherapy, and general physical condition, not by the results of HPV status and p16eviously . Each paThe cervical cancer cell lines CaSki (harboring ∼600 copies of integrated HPV-16 DNA/genome) and SiHa (1–2 copies of integrated HPV-16 DNA/genome) were purchased from the European Collection of Animal Cell Cultures  and the American Type Culture Collection , respectively, and cultured according to the suppliers’ instructions.™ kit , respectively, according to the manufacturers’ protocols. The extracted RNA was suspended in 50 μl ultra-high quality diethyl pyrocarbonate-treated water.Genomic DNA and total RNA were extracted from frozen tumor samples, SiHa cells and CaSki cells using the Gentra Purification Tissue kit  and the ToTALLY RNA+/GP6+ and MY09/11 (+/GP6+ or MY09/11 PCR were re-amplified by (auto-) nested PCR using the GP5+/GP6+ primer pair as previously described  according to the manufacturer’s instructions. To examine the presence of contaminating DNA in RNA samples, all the assays were performed both with and without reverse transcriptase.Before cDNA synthesis, any residual DNA was removed by incubation with 1 U DNase I (Ambion) at room temperature for 25 min. cDNA was then synthesized from DNA-free total RNA using the RETROscriptE6/E7 mRNA transcripts, PCR was performed with the cDNA from HPV DNA-positive samples using the Takara PCR Human Papillomavirus Typing Set , which can identify high-risk HPV types 16, 18, 31, 33, 35, 52 and 58. To verify the HPV-16 E6/E7 mRNA transcripts, the HPV-16 DNA-positive samples were also examined using a half-nested PCR approach with cDNA as previously described by Wiest et al . Endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide plus 15 mM sodium azide for 5 min. The sections were subsequently incubated overnight at 4°C with primary monoclonal mouse anti-p16INK4a antibody . After extensive washing in phosphate-buffered saline, the slides were incubated for 30 min at room temperature with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (MTM Laboratories). Immunolabeling was visualized by incubation in 3-3′-diaminobenzidine for 10 min. Stained slides were counterstained with hematoxylin.Serial 4-INK4a-positive when intense nuclear and/or cytoplasmic reactivity was present. The scoring criteria for p16INK4a immunoreactivity (p16INK4a expression) were defined for this study based on previous scoring methods  and regional lymph nodes (N site) after treatment. Recurrence-free survival was defined as the time from the end of treatment to cancer recurrence or last follow-up. Disease-specific survival was defined as the time from the end of treatment to subsidence of disease or last follow-up. Survival curves were evaluated by the Kaplan-Meier method, and survival distributions were compared using the log-rank test. Multivariate Cox proportional hazard analysis was used to identify prognostic parameters and treatments associated with risk of recurrence and disease-specific death. P-values <0.05 were considered significant. All statistical analyses were performed using the SPSS statistical package .Primary tumor location was the nasopharynx in 10 patients (6.7%), oropharynx in 53 (35.3%), hypopharynx in 39 (26.0%), larynx in 24 (16.0%) and oral cavity in 24 (16.0%). The follow-up period ranged from 6 to 77 months, with a median of 38 months for patients whose data were censored. Sixty-four patients were treated with CCRT, 45 with surgery and postoperative RT or CRT , 22 with surgery alone and 19 with RT alone. Demographic and clinical characteristics are summarized in HPV DNA was detected by PCR in 47 of 150 (31.3%) primary untreated HNSCC specimens, including 30.0% (3/10) of nasopharyngeal, 47.2% (25/53) of oropharyngeal , 17.9% (7/39) of hypopharyngeal, 16.7% (4/24) of laryngeal and 33.3% (8/24) of oral cavity cases. Among HPV-positive HNSCC samples, 39 (83.0 %) were infected with HPV-16 and the others were infected with other high-risk types .E6/E7 mRNA expression by HPV-16, HPV-33, HPV-35, HPV-58 and HPV-56 was examined by reverse transcription PCR in 45 samples. The E6 and E7 mRNA transcripts were detected in 21 of 45 (46.7%) specimens, the majority from OPSCC cases (18/25 HPV-positive cases), as shown in As two of these HPV-positive samples were insufficient for RNA assay, INK4a overexpression group showed similar clinical characteristics  was based on the percentage of p16INK4a-positive cells (INK4a was observed in 40 of 150 (26.7%) HNSCC samples  samples demonstrating a p16INK4a staining score ≥1. The sensitivity of p16INK4a staining for detection of HPV DNA in HNSCC was just 61.7%, as only 29 of 47 HPV-positive cases also had detectable p16INK4a staining. The specificity was 89.3% (92/103 HPV-negative samples had a p16INK4a staining score of 0) for all HNSCC cases , the inter-rater agreements were relatively low .In this study, the p16ve cells . Express samples . The higCC cases . AlthougINK4a expression, p16INK4a over-expression demonstrated high specificity for detection of HPV DNA in HNSCC (98/103 HPV-DNA negative cases did not overexpress p16INK4a) and OPSCC (28/28 cases). The sensitivity of p16INK4a overexpression was only 53.2% (25/47 cases) for detection of HPV DNA in HNSCC, but was considerably better for detection of HPV DNA in OPSCC at 80% (20/25 cases) (INK4a overexpression indicated the presence of HPV E6/E7 mRNA expression with high sensitivity at 90.5% (19/21) and high specificity at 91.3% (116/127) in HNSCC, and was even more accurate for OPSCC, with sensitivity at 94.4% (17/18) and specificity at 91.4% (32/35) (INK4a overexpression for HPV E6/E7 mRNA expression status was good for HNSCC (κ=0.69) and excellent for OPSCC (κ=0.84).In contrast to general p165 cases) . However (32/35) . MoreoveINK4a overexpression, subjects with OPSCC were divided into two groups: a double positive group of patients who were HPV DNA-positive with p16INK4a overexpression and a single positive-negative or double negative group of patients who were either HPV DNA-positive without p16INK4a overexpression, HPV DNA-negative with p16INK4a overexpression, or HPV-DNAnegative without p16INK4a overexpression. The combination of HPV DNA status and p16INK4a overexpression had both high sensitivity (94.4%) and specificity (100%) for detecting HPV E6/E7 mRNA expression in OPSCC  developed recurrent disease, including 2 of 10 (20.0%) patients with stage I/II OPSCC and 5 of 43 (11.6%) patients with advanced stage OPSCC. However, no patient died with disease during the follow-up period. Since the disease specific rate and overall survival rate in OPSCC were quite fair in the present study, the prognostic analysis of OPSCC focused on recurrence-free survival. Since there were few recurrence-free survival events in OPSCC cases, multivariate analysis of recurrence-free survival was carried out instead for HNSCC cases overall.E6/E7 mRNA expression and p16INK4a overexpression in OPSCC. HPV DNA-positive patients with OPSCC had better recurrence-free survival than HPV DNA-negative patients with OPSCC (P=0.008) (i) Prognostic value of HPV DNA status, P=0.008) . The recHPV mRNA-positive OPSCC patients exhibited a trend toward improved recurrence-free survival compared with HPV mRNA-negative patients with OPSCC (P=0.051) . The recINK4a overexpression showed significantly improved recurrence-free survival compared with OPSCC patients without p16INK4a overexpression (P=0.034) (OPSCC patients with p16P=0.034) . The recINK4a overexpression in OPSCC. Patients in the double positive group had better recurrence-free survival than those in a single negative groups or the double negative group (P=0.034) (ii) Comprehensive evaluation of survival according to HPV DNA status and p16P=0.034) , with reINK4a over-expression was modeled as one factor. Since no patients with positive HPV E6/E7 mRNA expression had any recurrent lesion, the influence of HPV mRNA expression on recurrence-free survival could not be evaluated.iii) Multivariate analysis using Cox proportional-hazard model in HNSCC. To assess the independent predictive value of all these factors for recurrence-free survival in HNSCC, multivariate analysis using Cox proportional-hazards models was performed. Both HPV DNA presence and p16INK4a overexpression demonstrated significantly higher recurrence-free survival  = 8.61; 95% confidence interval (CI) = 1.12–66.18) compared with other HNSCC patients  predicted significantly better recurrence-free survival  and the combination of HPV DNA-positive status and p16survival .Over the past two decades, HR-HPV has been firmly established as a common etiologic factor in OPSCC and is now widely used as a prognostic marker for OPSCC. Although there are a number of studies on the epidemiologic role and prognostic value of HPV in OPSCC, some did not distinguish between patients receiving curative treatment from those receiving palliative care –31. Moreet al systematically reviewed the published data regarding the prognostic significance of HPV in SCCs of the oral cavity, larynx, sinonasal tract and nasopharynx and found no association between HPV status and treatment outcome  also displayed improved recurrence-free survival compared with OPSCC patients without HPV mRNA expression (P=0.051), in line with previous studies ,9,32–35. outcome . FurtherINK4a expression can be used as a surrogate marker for HPV infection in OPSCC  and OPSCC (κ=0.57). These results indicate that p16INK4a expression alone is not suitable for identifying HPV-related tumors. However, p16INK4a over-expression (p16INK4a expression score ≥3) was a sensitive and specific marker for detecting HR-HPV mRNA expression in both HNSCC and OPSCC. This result also underscores the potential of our scoring system for evaluating p16INK4a expression and determining prognosis.In the present study, the sensitivity of general p16INK4a expression as a surrogate marker of HPV infection and fair prognosis in OPSCC. Lassen et al reported that p16INK4a-positive HNSCC showed a better response to conventional radiotherapy than p16INK4a-negative HNSCC, and ascribed this survival benefit to a better locoregional control rate  and sensitivity (94.4%). Furthermore, the combined evaluation of HPV DNA status and p16INK4a overexpression was an independent prognostic indicator for HNSCC in multivariate analysis. Given the time and expense of E6/E7 mRNA analysis, this combination may be particularly useful for larger scale clinical studies.Recent studies have demonstrated a significant correlation between p16rol rate . In a ste (et al , p16INK4modality . In the INK4a overexpression was strongly correlated with HPV mRNA expression in OPSCC. Since the combination of HPV DNA-positive status and p16INK4a overexpression showed a close relation with fair recurrence-free survival in HNSCC in multivariate analysis, the combination can serve as an accurate surrogate marker for biologically active HPV infection. This combined evaluation appears to be a useful and reliable method for detecting HPV-related HNSCC and determining its prognosis.The combined evaluation of HPV DNA status and p16


Women who were high school graduates/some college  and those with at least an associate degree  were more likely to be aware of the HPV vaccine than women without a high school degree. Our study documents poor knowledge of HPV and HPV vaccine in this population of IBD patients in Puerto Rico. Although vaccination coverage is low in this population, women are receptive to the possibility of vaccination in the future. Given that this population may be at an increased risk of HPV infection and related morbidities, education and vaccination programs should be promoted among them.Women with inflammatory bowel disease (IBD) may be at increased risk of human papilloma virus (HPV) infection and HPV-related malignancies, as many are immunocompromised secondary to the use of immunosuppressant agents. Several studies have addressed the knowledge about cervical cancer risk factors in different populations, particularly HPV infection and its association with cervical malignancies; most of these studies show poor patient knowledge. The purpose of this study is to describe the knowledge of females with IBD about HPV infection and the HPV vaccine. We performed a cross-sectional study in 147 consecutive patients attending the clinics of the University of Puerto Rico Center for IBD from 2009 to 2010. An interviewer-administered questionnaire was used to collect information on demographics, lifestyles, and HPV-related knowledge of participants. Bivariate analysis using the chi-square statistics and Fisher’s exact test was used to examine factors associated with HPV awareness. The mean age of participants was 36.6 years (SD = 13.91 years). Three fourth (77 %) of women had awareness of the existence of HPV, and 58 % did know about the existence of HPV vaccines. Among those who had heard about HPV, 79.6 % knew that HPV can cause cervical cancer, and 57.5 % knew that the virus is sexually transmitted. Among those who knew of the vaccine, 75.3 % learned about its existence through the media, while only 15.3 %, through their health-care provider. Only three women within recommended ages (2 %) had been vaccinated against HPV, although 50 % of participants indicated that they would definitely/probably vaccinate against HPV in the future. A significant trend was observed, where more educated women were more likely to have heard of HPV ( Human papilloma virus (HPV) is the primary etiologic agent for the development of cervical cancer and cervical dysplasia, along with other premalignant and malignant conditions of the vagina, vulva, and cervix. HPV has been also associated to anorectal, oropharyngeal, and head and neck cancer. It is estimated that 20 million Americans are infected with this virus, and a recent study has calculated the prevalence in women aged 14–59 years at 42.5 %  comparedCervical cancer has a major impact in females, particularly in developing countries where it is one of the leading causes of cancer death annually. According to recent estimates, 530,000 new cases of cervical cancer occur each year in women and 275,000 died of this disease in 2008 . Latin ASeveral studies have demonstrated poor knowledge about HPV and its association with cervical cancer in the Hispanic population –10. HispIn view of the close medical follow-up demanded for patient with chronic inflammatory conditions frequently requiring immunosuppressive medication, it would be expected that young women with IBD were more aware of routine health maintenance medical care, such as HPV vaccination and cervical cancer prevention. This study describes the knowledge about HPV infection and cervical cancer risk factors in a cohort of Hispanic women with IBD. Factors associated with HPV awareness and HPV vaccine awareness were analyzed.This cross-sectional descriptive study was performed at the UPR Center for Inflammatory Bowel Diseases clinics from February 2009 until June 2010. The study had approval from the University of Puerto Rico Medical Sciences Campus Institutional Review Board. The research coordinator identified potential participants among patients attending the clinics. All women older than 15 years with established diagnosis of IBD evaluated at the clinic were invited to participate in the study. From approximately 170 subjects invited to participate, 147 (86.5 %) women agreed to participate and were recruited. All study participants provided informed consent at the clinic. For subjects under 21 years old, informed consent was obtained from both the subject and the parents or legal guardian. After consent, research personnel administered a survey to the study participants at the clinic. The survey was administered in Spanish, the native language of all the participants. Estimated time of completion of the survey was 25 min. Surveys were identified with a code to protect confidentiality. The survey included questions about demographic information , lifestyle (use of tobacco), medical history , reproductive health , sexual practices , knowledge about HPV and other sexually transmitted diseases (STDs), and HPV vaccination history . In the education groups, participants were classified into those who did not complete high school education (no high school degree), those who completed high school but did not complete a college degree (high school graduate/no college degree), and those who obtained a college degree and/or completed postgraduate education (college degree or above). Civil status was classified as those who reported being single and in no romantic relationship, those married or in a romantic relationship, and those divorced or widowed. HPV awareness was evaluated with questions as follows: ‘Have you ever heard about the Human Papilloma Virus?’ (yes or no). HPV vaccine awareness was evaluated with the following question: ‘Have you ever heard about the existence of a Human Papilloma Virus vaccine?’ (yes or no). Other statements that reflected knowledge about symptoms of infection with HPV or cervical cancer and lifestyles that may increase the risk of HPV infection were also evaluated. This survey is a modification of a survey used by Colón-Lopez et al. to assess HPV awareness and knowledge on a Hispanic population .Data from the study was recorded in a database designed with the use of Epi Info™, version 3.3.2. . Statistn = 113) answered general questions about HPV knowledge and risk factors. Among those who had ever heard about HPV, 79.6 % knew that HPV can cause cervical cancer and 57.5 % knew that the virus is sexually transmitted. Only 31.0 % knew that genital warts are caused by HPV while 19.5 % of the participants erroneously associated genital warts with herpes virus. A considerable percentage of the participants knew about risk factors for HPV infection. However, only half of the participants recognized that first coitus before the age of 16 years is an important risk factor for HPV infection. Most of the participants identified having multiple sexual partners and unprotected sex as important risk factors.The mean age of participants was 36.6 years SD = 13.91 year). Demographic characteristics are outlined in Table 3.91 yearp < 0.05).Table Among those who knew of the vaccine, 75 % learned about its existence through the media, while only 17 % through their physician/health care professional. Only 3 women of the 147 participants (2 %) had been vaccinated against HPV. This represents only 5 % compliance (three of 56 participants who were 9–26 years old) with the recommendations of the Advisory Committee for Immunization Practices [p = 0.005 and p = 0.0002, respectively). No other covariate was significantly (p < 0.05) associated with HPV and HPV vaccine awareness in bivariate analysis. In age-adjusted logistic regression analysis (Table p for trend = 0.0017). Women who were high school graduates/some college  and those with at least an associate degree  were more likely to be aware of the HPV vaccine than women without a high school degree. No significant interaction was observed between age and educational attainment within the model .Table is Table , a signiPatients with chronic conditions requiring immunosuppressive therapy, such as a patient with IBD, need a more structured and regular follow-up by medical providers to address different special health needs. The comprehensive management of these patients includes special attention to routine health management recommendations like vaccination. In IBD patients, HPV vaccine should be encouraged in view of the foreseeable risk of cervical cancer and the proven benefits of vaccination. It would be expected that young women with IBD were more aware of routine health maintenance medical care, like HPV vaccination and cervical cancer prevention. However, this study demonstrates low awareness about HPV and HPV vaccine in women with IBD evaluated in the UPR Center for Inflammatory Bowel Diseases. These findings are comparable with other studies in Hispanic populations. A study performed by Tortolero et al. found similar levels of awareness of HPV in Puerto Rican women over 18 years old . Despitep = 0.005). Regression analysis in our study demonstrated that younger women had higher awareness of HPV and HPV vaccine. In a similar study performed by Drewry et al. among Latina immigrants aged 19 to 50 years old, 47 % of participants reported being aware of HPV [The finding of poor knowledge about HPV in Hispanics or Latina women has been documented in several studies. For example, a study published by Markham et al. in Latino youth enrolled in nine alternative high schools in Houston, Texas, found that HPV knowledge was significantly lower in youths whose mothers were born outside USA versus youths of USA-born mothers . In our e of HPV . This ste of HPV . Unfortue of HPV  describee of HPV .Several factors may explain the low vaccination compliance in our high-risk IBD population: insurance coverage, poor referral to vaccination programs, and limited access to vaccination programs. One of the financial barriers to widespread vaccination is that health insurance coverage of vaccination is not universal in Puerto Rico. Vaccination programs do not sponsor vaccination for patients 19–26 years old, and not all private insurances cover vaccination for patients in this same age range. Also, it is our impression that few primary providers have the infrastructure to offer vaccination in their clinics. This might be related to the increasing cost of vaccines and more strict and costly regulations required of providers, vaccination centers, and programs. Under these circumstances, appropriate referral and increasing awareness of vaccination in this population should be underscored. In our study, from 140 participants who reported reasons for not being vaccinated, almost 40 % were not aware of the existence of the HPV vaccine, 10.7 % considered not being at risk of HPV, and 5.0 % were worried of possible side effects. Teitelman et al. identified several barriers for HPV vaccination in a group of young women aged 13 to 26 years from an economically disadvantaged urban community . Among tThis study has some limitations. This is a cross-sectional study, and no causal relationship can be established in terms of knowledge of HPV and cervical cancer risk factors and the actual risk of HPV infection and cervical cancer risk. This study included a relatively small sample of patients limiting the generalization of findings obtained in subgroup analysis. This specific limitation might be addressed in a well-designed population-based study. We had limited gynecologic information of study participants and were unable to establish the prevalence of HPV infection and cervico-vaginal abnormalities in the women that visited our clinic. In addition, we were unable to include a healthy control group to compare the findings with those of the IBD patients. However, a similar study has been performed in Puerto Rican women without IBD .Awareness of HPV risk factors and HPV vaccination is unsatisfactory in this underserved population. Our study illustrates this medical issue in women with IBD evaluated in the UPR Center for Inflammatory Bowel Diseases. There is a need to develop education and vaccination programs that can directly impact this high-risk population; targeted education efforts should focus on women with reduced educational attainment. Physicians and health-care workers who care for IBD patients should encourage vaccination, preferably before administration of immunosuppressive medications. Also, it is important that physicians and health-care workers who care for women with IBD encourage compliance with current endorsed cervico-vaginal cancer surveillance recommendations, based on age and risk factors. This is particularly important for gastroenterologists closely involved in the treatment of IBD. Despite the paucity of scientific data about the most appropriate surveillance in women with IBD using immunosuppressive medication, it is our opinion that this group should be considered at high risk for malignancy. Furthermore, cervical cancer surveillance may be more important in women older than 26, with no previous history of HPV immunization, who may have more cumulative exposure to immunosuppressive drugs in view of length of disease and in whom vaccination is currently not endorsed by current clinical guidelines. How cost-effective might vaccination be in women with IBD older than 26 years old is an undefined topic.