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Sotirios G Papageorgiou,1 Aspasia Divane,2 Maria Roumelioti,3 Christine Kottaridi,4 Anthi Bouchla,1 Alexandros Georgakopoulos,5 Fotini Ieremiadou,2 Aggeliki Daraki,2 Efthymia Bazani,1 Thomas P Thomopoulos,1 Sofia Chatziioannou,5,6 Andreas Mavrogenis,7 Panayiotis Panayiotidis,3 Ioannis G Panayiotides,4 Vasiliki Pappa,1,* Periklis G Foukas4,* 1 2nd Department of Internal Medicine and Research Unit, Hematology Unit, University General Hospital “Attikon”, Haidari, Athens, Greece; 2“LIFE CODE” Private Diagnostic Laboratory, Medical Ltd., Athens, Greece; 3 1st Department of Propaedeutic Medicine, Laikon General Hospital, National and Kapodistrian University of Athens, Athens, Greece; 4 2nd Department of Pathology, National and Kapodistrian University of Athens, Medical School, University General Hospital “Attikon”, Haidari, Athens, Greece; 5 2nd Department of Radiology, Nuclear Medicine Section, National and Kapodistrian University of Athens, University General Hospital “Attikon”, Haidari, Athens, Greece; 6Nuclear Medicine Section, Biomedical Research Foundation Academy of Athens, BRFAA, Athens, Greece; 7 1st Department of Orthopaedics, National and Kapodistrian University of Athens, School of Medicine, University General Hospital “Attikon”, Haidari, Athens, Greece*These authors contributed equally to this workCorrespondence: Sotirios G Papageorgiou 2nd Department of Internal Medicine and Research Unit, University General Hospital “Attikon”, 1 Rimini St., Haidari 12462 Athens, GreeceTel +30 210-583-2318Fax +30 210-538-2306Email sotirispapageorgiou@hotmail.comBackground: Erdheim–Chester Disease (ECD) is a clonal non-Langerhans histiocytosis, classified as a macrophage-dendritic cell neoplasm in the 2016 WHO classification. The exact cell of origin of ECD is unknown, although some limited evidence suggests that it arises from myeloid progenitors.Case Presentation: A 43-year-old patient, diagnosed with BRAFV600E mutated ECD, developed NPM1+/FLT3+ acute myeloid leukemia (AML) with wild-type BRAF, 15 months after the initial ECD diagnosis. The patient received intensive chemotherapy plus midostaurin, followed by midostaurin maintenance. Six months into maintenance, the patient remains in complete remission with low-level measurable residual disease, whereas ECD shows a sustained partial metabolic response. Molecular karyotype at several distinct timepoints, namely ECD diagnosis, AML diagnosis, and following treatment of AML, highlighted a molecular signature, indicative of a persistent, underlying clonal hematopoiesis.Conclusion: This case report suggests that ECD and AML might represent an expansion of two distinct clones in a background of clonal hematopoiesis, indicating their shared origin. Moreover, molecular karyotype might serve as a strong, inexpensive tool for revealing clonal hematopoiesis in cases of negative targeted next-generation sequencing. Finally, the moderate response of ECD to midostaurin suggests that kinase inhibition might have a potential role in ECD treatment.Keywords: Erdheim–Chester disease, acute myeloid leukemia, clonal hematopoiesis, case report, molecular karyotype, midostaurin

HPV16 infection is found in more than 50 % of cervical cancer cases worldwide, triggering the development of numerous molecular techniques for viral diagnosis. The present study focuses on the development of a colorimetric IsoPCR for HPV16 DNA detection. The methodology combines the advantages of PCR and LAMP, while the most significant aspect of the new established methodology is the visual detection of amplification products through hydroxynapthol blue dye, thus minimizing the time and labor needed. An experimental cut-off value was tested through reconstitution experiments, while the specificity was evaluated by assessing clinical samples. The analytical sensitivity of the new colorimetric IsoPCR was found to be 0.1 viral DNA copy per reaction, while the specificity was 100 % for the detection of HPV16 DNA. The assay enabled the amplification of viral DNA in cases with viral load lower than 1 copy. In conclusion, the new established colorimetric IsoPCR can be regarded as an attractive molecular tool that facilitates the specific, rapid and highly sensitive visual detection of HPV16 DNA even at the very early stages of viral infection. © 2021 Elsevier B.V.

Background: Erdheim–Chester Disease (ECD) is a clonal non-Langerhans histiocytosis, classified as a macrophage-dendritic cell neoplasm in the 2016 WHO classification. The exact cell of origin of ECD is unknown, although some limited evidence suggests that it arises from myeloid progenitors. Case Presentation: A 43-year-old patient, diagnosed with BRAFV600E mutated ECD, developed NPM1+/FLT3+ acute myeloid leukemia (AML) with wild-type BRAF, 15 months after the initial ECD diagnosis. The patient received intensive chemotherapy plus midos-taurin, followed by midostaurin maintenance. Six months into maintenance, the patient remains in complete remission with low-level measurable residual disease, whereas ECD shows a sustained partial metabolic response. Molecular karyotype at several distinct time-points, namely ECD diagnosis, AML diagnosis, and following treatment of AML, high-lighted a molecular signature, indicative of a persistent, underlying clonal hematopoiesis. Conclusion: This case report suggests that ECD and AML might represent an expansion of two distinct clones in a background of clonal hematopoiesis, indicating their shared origin. Moreover, molecular karyotype might serve as a strong, inexpensive tool for revealing clonal hematopoiesis in cases of negative targeted next-generation sequencing. Finally, the moder-ate response of ECD to midostaurin suggests that kinase inhibition might have a potential role in ECD treatment. © 2020 Papageorgiou et al.

Introduction: Short telomeres are recognized as risk factor for idiopathic pulmonary fibrosis (IPF). We aimed to assess the role of telomere length (TL) in fibrotic-Interstitial Lung Diseases (f-ILDs) associated with a usual interstitial pneumonia (UIP) pattern as well as in IPF acute exacerbation (IPF-AE). Aim and methods: TL was measured from peripheral white blood cells using a multiplex quantitative polymerase chain reaction in consecutive patients with f-ILDs, all presenting UIP pattern in the high-resolution chest-computed-tomography and compared to age-matched healthy controls. Results: Seventy-nine individuals were included (mean age 69.77 ± 0.72 years); 24 stable IPF, 18 IPF-AE, 10 combined pulmonary fibrosis and emphysema, 7 Rheumatoid arthritis-UIP-ILDs and 20 controls. TL in all patients was significantly shorter compared to controls [mean T/S ratio (SE) 0.77 (±0.05) vs 2.26 (±0.36), p < 0.001] as well as separately in each one of f-ILD subgroups. IPF-AE patients presented significantly shorter TL compared to stable IPF (p = 0.029). Patients with IPF and shorter than the median TL (0−0.72) showed reduced overall survival (p = 0.004). T/S < 0.72 was associated with increased risk for IPF-AE (OR = 30.787, 95% CI: 2.153, 440.183, p = 0.012) independent of age, gender, smoking and lung function impairment. A protective effect of TL was observed, as it was inversely associated with risk of death both in UIP-f-ILDs (HR = 0.174, 95%CI: 0.036, 0.846, p = 0.030) and IPF patients (HR = 0.096, 95%CI: 0.011, 0.849, p = 0.035). Conclusions: Shorter TL characterizes different UIP f-ILDs. Although no difference was observed in TL among diverse UIP subgroups, IPF-AE presented shorter TL compared to stable IPF. Reduced overall survival and higher hazard ratio of death are associated with shorter TL in IPF. © 2020 Sociedade Portuguesa de Pneumologia

The objective of this study was to investigate the hypothesis that HPV vaccination administered in patients with low-grade (LG) cytology shortly after an initial colposcopic assessment could prospectively alter HPV-related biomarkers. This was a prospective pilot observational study involving women attending a colposcopy clinic for evaluation of abnormal LG cytology that were advised to undergo HPV vaccination and proceeded accordingly. These women were compared with a matched unvaccinated group. Women requiring cervical biopsies or CIN treatment were excluded. Intervention: A full three-dose HPV vaccination was undertaken with either the 2-valent or the 4-valent anti-HPV VLP vaccine. LBC samples were obtained prior and after the completion of the vaccination regimen and tested for HPV DNA genotyping (CLART-2 HPV test) and E6 and E7 mRNA (NASBA technique). Results: Alterations of HPV-related biomarkers at a colposcopy reassessment appointment 12 months later. Analysis: The p-values, relative risk (RR), absolute relative risk (ARR), number needed to treat (NNT) and 95% confidence intervals for each biomarker in each group were assessed. Results: A total of 309 women were included in the analysis. One hundred fifty-two women received the vaccine. HPV vaccination reduced in a statistically significant manner (p < 0.05) HPV DNA positivity rates for genotypes 16, 18, and 31, RR = 1.6 (95% CI: 1.1 to 2.3), RR = 1.7 (95% CI: 1.1 to 2.8), and RR = 1.8 (95% CI: 1.0 to 2.9), in women who only tested DNA-positive for HPV16, 18, and 31 genotypes, respectively, prior to vaccination. A less pronounced, statistically insignificant reduction was shown for women who tested positive for both HPV DNA and mRNA E6 and E7 expression for HPV16, 18, and 33 subtypes. Statistically significant reduction in HPV mRNA positivity was solely documented for genotype 31 (p = 0.0411). Conclusions: HPV vaccination appears to significantly affect the rates of HPV16, 18, and 31 DNA-positive infections in the population testing HPV DNA-positive for the aforementioned genotypes. The above findings deserve verification in larger cohorts. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Aim and Methods: Dried blood spots from 2149 newborns were examined to diagnose congenital cytomegalovirus (cCMV). Results: Prenatal CMV-IgG antibodies had been measured during prenatal care in 1287 (60.3%) of mothers and 980 (76.1%) of them were found seropositive. cCMV incidence was 0.47%. All newborns were asymptomatic; 9/10 were born post nonprimary maternal infection; two developed sensorineural hearing loss. Conclusions: In a country where prenatal CMV testing is common and therefore a false sense of control might prevail, nonprimary maternal infection should not be overlooked. Indeed, women of childbearing age should be educated on CMV prevention measures irrespectively to their serostatus. © 2017, © 2017 Informa UK Limited, trading as Taylor & Francis Group.

Background: Standardised clinical outcome measures are urgently needed for the surveillance of influenza and influenza-like illness (ILI) based on individual patient data (IPD). Objectives: We report a multicentre prospective cohort using a predefined disease severity score in routine care. Patients/Methods: The Vienna Vaccine Safety initiative (ViVI) Disease Severity Score (“ViVI Score”) was made available as an android-based mobile application to three paediatric hospitals in Berlin and Athens between 2013 and 2016. Healthcare professionals assessed ILI patients at the point of care including severity, risk factors and use of antibiotics/antivirals/vaccines. RT-PCR for influenza A/B viruses was performed at the Hellenic Pasteur Institute and the Robert Koch Institute. PCR testing was blinded to severity scoring and vice versa. Results: A total of 1615 children aged 0-5 years (54.4% males) were assessed at the three sites. The mean age was 1.7 years (SD 1.5; range 0-5.9). The success rate (completion of the scoring without disruption to the ER workflow) was 100%. ViVI Disease Severity Scores ranged from 0 to 35 (mean 13.72). Disease severity in the Berlin Cohort was slightly higher (mean 15.26) compared to the Athens Cohorts (mean 10.86 and 11.13). The administration of antibiotics was most prevalent in the Berlin Cohort, with 41.2% on antibiotics (predominantly cefuroxime) as opposed to only 0.5% on neuraminidase inhibitors. Overall, Risk-adjusted ViVI Scores were significantly linked to the prescription of both, antibiotics and antivirals. Conclusions: The Risk-adjusted ViVI Score enables a precision medicine approach to managing ILI in multicentre settings. Using mobile applications, severity data will be obtained in real time with important implications for the evaluation of antiviral/vaccine use. © 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Aim: Limited data are available regarding the clinical relevance of platelet function measurements in stable patients with coronary artery disease (CAD). Our aim is to evaluate the agreement between multiple electrode aggregometry (MEA) and light transmittance aggregometry (LTA) in detecting clopidogrel low responders and their prognostic value in CAD patients with type 2 diabetes mellitus (T2DM) on dual platelet inhibition. Methods: LTA and MEA were performed in 122 stable cardiovascular patients with T2DM. The upper quartile of patients according to maximum LTA (LTAmax) and MEA measurements were defined as clopidogrel low responders. Agreement between the two methods was evaluated by kappa statistics. We assessed the potential correlation between antiplatelet response and clinical outcome and the optimal cutoff value according to ROC analysis to predict the occurrence of major adverse cardiovascular events (MACE), during 1-year follow-up period. Results: Cohen's kappa coefficients (0.214) indicated fair agreement (70.2%) between LTA and MEA. A total of 25 MACE occurred in 108 patients (23.1%). Patients with MACE had higher LTAmax than those without (57.1 ± 16.5 vs 49.3 ± 18.3, respectively, p = 0.023). MEA measurements were similar between patients with and without MACE (30.1 ± 15.4 vs 30.6 ± 20.8, respectively; p = 0.84). Multiple logistic regression showed LTAmax response as an independent predictor of death from cardiovascular causes (Odds Ratio, adjusted:0.2;0.05–0.81). ROC analysis indicated that LTAmax cutoff of 62.5% best predicted death (AUC = 0.67, sensitivity = 78%, specificity = 61.5%). Conclusions: The assessment of platelet responsiveness remains highly test-specific. Our results support the prognostic role of LTA, but not MEA testing, for death risk evaluation in stable cardiovascular T2DM patients. © 2019 Elsevier Ltd

Background: Human Papilloma Virus has been considered as the main cause for cervical cancer. In this study we investigated epigenetic changes and especially methylation of specific sites of HPV genome. The main goal was to correlate methylation status with histological grade as well as to determine its accuracy in predicting the disease severity by establishing optimum methylation cutoffs. Methods: In total, sections from 145 cases genotyped as HPV16 were obtained from formalin-fixed, paraffin-embedded tissue of cervical biopsies, conization or hysterectomy specimens. Highly accurate pyrosequencing of bisulfite converted DNA, was used to quantify the methylation percentages of UTR promoter, enhancer and 5’ UTR, E6 CpGs 494, 502, 506 and E7 CpGs 765, 780, 790. The samples were separated in different groupings based on the histological outcome. Statistical analysis was performed by SAS 9.4 for Windows and methylation cutoffs were identified by MATLAB programming language. Results: The most important methylation sites were at the enhancer and especially UTR 7535 and 7553 sites. Specifically for CIN3+ (i.e. HSIL or SCC) discrimination, a balanced sensitivity vs. specificity (68.1%, 66.2% respectively) with positive predictive value (PPV) and negative predictive value (NPV) (66.2%, 68.2% respectively) was achieved for UTR 7535 methylation of 6.1% cutoff with overall accuracy 67.1%, while for UTR 7553 a sensitivity 60.9%, specificity 69.0%, PPV=65.6%, NPV=64.5% and overall accuracy=65.0% at threshold 10.1% was observed. Conclusion: Viral HPV16 genome was found methylated in NF-1 binding sites of UTR in cases with high grade disease. Methylation percentages of E6 and E7 CpG sites were elevated at the cancer group. © The author(s).

Respiratory infections in oncology are both common and potentially severe. However, there is still a gap in the literature, regarding the epidemiology of viral respiratory infections in children with cancer. We prospectively enrolled 224 patients, from September 2012 to August 2015. The cohort included children with hematologic or solid malignancies receiving chemotherapy, or undergoing hemopoietic stem cell transplantation, outpatients/inpatients exhibiting signs/symptoms of febrile/afebrile upper/lower respiratory infection. Viral infection was diagnosed by detection of ≥1 viruses from a sample at time of enrollment, using the CLART® PneumoVir kit (GENOMICA, Spain). Α detailed questionnaire including demographics and medical history was also completed. Samples were processed in batches, results were communicated as soon as they became available. Children recruited in whom no virus was detected composed the no virus detected group. Viral prevalence was 38.4% in children presenting with respiratory illness. A single virus was found in 30.4%, with RSV being the most frequent. Viral coinfections were detected in 8%. Children with viral infection were more likely to be febrile upon enrollment and to present with lower respiratory signs/symptoms. They had longer duration of illness and they were more likely to receive antibiotics/antifungals. Only 22% of children with influenza received oseltamivir. Mortality was low (2.7%), however, pediatric intensive care unit (PICU) admission and death were correlated with virus detection. In our study mortality was low and PICU admission was related to virus identification. Further research is needed to clarify whether antibiotics in virus-proven infection are of value and underline the importance of oseltamivir’s timely administration in influenza. © 2019, © 2019 Taylor & Francis Group, LLC.

Background and objectives: Persistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. Methods: The method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. Results: The WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3)for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. Conclusions: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes. © 2019 Elsevier B.V.

Aim: To prospectively evaluate interferences between viruses of the upper respiratory tract in asymptomatic preschool children. Methods: Nasal-pharyngeal swabs from 233 preschool aged children were prospectively collected over four consecutive time periods, during one school year. The samples were tested using a RT-PCR DNA/RNA microarray system for nine respiratory viruses. Results: Respiratory syncytial virus (RSV) was a predictor of the presence of influenza virus (INFL) (OR: 9.12, CI: 1.52–54.75, p = 0.016), and similarly, INFL predicted the presence of RSV (OR: 4.01, CI: 1.14–14.16, p = 0.030). Also, rhinovirus (RV) was a predictor of adenovirus (ADV) presence (OR: 3.66, CI: 1.10–12.14, p = 0.034), and similarly, ADV predicted the presence of RV (OR: 4.05, CI: 1.02–16.05, p = 0.046). No other significant associations between viruses were observed. Conclusion: Our results indicate that respiratory viruses found in carrier stage in asymptomatic children may interact with other viruses and even facilitate their settling in the upper respiratory tract. The pathophysiological role of these interactions is not yet clear. © 2018 SEICAP

Molecular detection of HPV DNA is considered as the gold standard for the diagnosis of cervical disease. Although the molecular assays for the identification of HPV16 and HPV18 have helped identify cervical cancer incidents, they are restricted to specialized laboratories. Thus, we developed a novel 2-stage, nested-like nucleic acid amplification method, named IsoPCR, to amplify the E6 gene of HPV16 and HPV18 with high analytical sensitivity and specificity. The performance of IsoPCR was compared to that of conventional PCR and LAMP. The analytical sensitivity of IsoPCR (1 copy/test)was 10-fold higher than conventional PCR and 25-fold higher than conventional LAMP. IsoPCR displayed significant amplification specificity (100%)and efficiency, as well. In conclusion, IsoPCR is a highly sensitive and specific diagnostic tool and it is suitable for the detection of low copy number of viral DNA in clinical specimens, providing critical information to healthcare providers. © 2019 Elsevier Ltd

Background: Flow cytometry (FC) and Nageotte hemocytometry represent the most widely accepted methods for counting residual white blood cells (rWBCs) in leucocyte-reduced (LR) blood components. Our aim was to study the agreement between the two methods, under real working blood bank conditions. Materials and methods: 94 freshly produced LR red blood cell (RBC) units were tested for rWBC concentrations by FC and Nageotte. To assess the precision of each method, we calculated the intra-assay coefficients of variation (CV), and followed the Bland-Altman methodology to study the agreement between the two methods. Results: CV was 18.5% and 26.2% for the Nageotte and the FC, respectively. However, the agreement between the duplicate observations, using the binary cut-off threshold of 1 × 106 WBCs per unit to define the results as “pass/fail” was 71.9% for the Nageotte and 93.3% for the FC. Linear regression analysis did not show any correlation (R-squared = 0.01, p = 0.35) between the two methods, while the Bland-Altman analysis for the measuring agreement showed a bias toward a higher Nageotte count of 0.77 × 106 leucocytes per unit (p < 0.001) with the 95% limits of agreement (d ± 2 sd) ranging from –0.40 × 106 to 1.94 × 106 leucocytes per unit. Conclusion: The absence of agreement between Nageotte and FC method, with the differences within d ± 2 sd being of high clinical importance, suggests that the two methods cannot be used for clinical purposes interchangeably. The Nageotte seems unsuitable for quality control even with a pass-fail criterion, under real working blood bank conditions. © 2018 Elsevier Ltd

The current study aimed to describe the molecular epidemiology of mixed respiratory viral infections during consecutive winter seasons in a tertiary care hospital. Patients with symptoms of respiratory tract infection were evaluated during the 2009-2011 and 2013-15 winter seasons. A clinical microarray technique was used for viral detection. Clinical and epidemiological data were correlated with mixed viral detection and the need for hospitalization. In 332 out of 604 (54.4%) evaluated patients (17.6% children) a respiratory virus was identified. Mixed viral infections were diagnosed in 68/332 (20.5%) patients with virus detection (66.2% mixed Influenza-RSV infections). Mixed viral infections were more commonly detected in children (OR 3.7; 95%CI 1.9-5.6, P < 0.01) and patients with comorbidities. In logistic regression analyses, mixed viral infections were associated with younger age (mean age 30.4 years vs. 41.8 years, P ≤ 0.001) and increased rates of fever (OR: 2.7; 95%CI 1.04-7.2, P < 0.05) but no adverse outcomes or increased rates of hospitalization. High rates of mixed viral infections were noted during all winter seasons (especially Influenza and RSV) and were more common in younger patients. The clinical significance of mixed respiratory viral infection needs further elucidation. © 2017 Wiley Periodicals, Inc.

Purpose. The tumour suppressor protein RB plays a decisive role in negative control of the cell cycle, inhibiting tumour development. The present analysis investigated the prevalence of the nucleotide polymorphism A153104G, which is located at intron 18 of the RB1 gene, and investigated the impact of the polymorphic variability in the exon 19 and its flanking intronic sequences on the severity of cervical disease in HPV16-positive Greek women. Methodology. The nucleotide polymorphism A153104G was detected by PCR-RFLP assay, while the amplicons were further subjected to cloning and sequencing. Moreover, molecular evolutionary analysis was performed using the maximumlikelihood (ML) and empirical Bayesian (EB) methods in order to evaluate the selective pressure acting on exon 19 of the RB1 gene. Results/Key findings. The A153104G nucleotide polymorphism was only detected in one control case. Moreover, sequence analysis of the amplicons revealed that the polymorphic variability in the RB1 gene increased with the severity of the cervical dysplasia. The link between the observed polymorphic variability and the progress of cervical disease was reflected in the molecular evolutionary analysis that was performed on the exon 19 of the RB1 gene, since negative selective pressure was acting upon exon 19 in the control and low-grade squamous intraepithelial lesion (LSIL) cervical samples, while positive selective pressure was acting upon exon 19 in the high-grade squamous intraepithelial lesion (HSIL) specimens. Conclusions. The A153104G nucleotide polymorphism did not emerge as a potential biomarker for the development of precancerous lesions in the Greek patients, while the accumulation of sequence variations in RB1 gene might influence patients’ susceptibility towards the progression of cervical neoplasia. © 2018 The Authors.

The tumor suppressor protein p16 plays a fundamental role in cell cycle regulation and exerts a protective effect against tumor growth. Two different polymorphisms at positions 540 and 580 at the 3′UTR of exon 3 of p16 gene are implicated in several types of cancer, while their role in cervical cancer development remains rather vague. In the present study, we investigated for the impact of p16 genotypes/haplotypes on patients' vulnerability to cervical disease and examined whether these factors can be used as progression markers in the Greek population. A total of 96 HPV16 positive samples and histologically confirmed as LSIL (42 samples), HSIL (44 samples), and cervical cancer cases (10 samples) along with 50 control cases were tested. The identification of p16 polymorphisms was performed by PCR-RFLP methodology. The present analysis revealed that women with p16 540 CG/GG genotype are at a 2.7-fold higher risk of developing HPV16-associated HSIL (OR = 2.7, 95%CI: 1.01-6.6, P = 0.028). The G allele can be regarded as a risk factor of developing HSIL in the Greek population (OR = 2.7, 95%CI: 1.2-5.9, P = 0.012). Moreover, p16 polymorphism C580T is not associated with the growth of cervical lesion in Greek patients, while 540G/580C haplotype can be regarded as a risk haplotype of developing HSIL (OR = 3.67, 95%CI: 1.56-8.6, P = 0.0019). Our results demonstrated that p16 C540G polymorphism influence patients' susceptibility to more severe dysplasia and consequently this polymorphism could potentially emerge as a valuable biomarker for HSIL development in the Greek population. © 2017 Wiley Periodicals, Inc.

Background: miRNAs have an important role as their deregulation is linked to endometrial cancer. Methods: A custom miScript® miRNA PCR Array was used to investigate for the first time the expression of eight miRNAs in forty-nine histologically confirmed Liquid Based cytology endometrial samples. The expression profile of the same miRNAs was also examined in sixty formalin-fixed tissue samples. Results: Expression of seven miRNAs was significantly higher in malignant samples with three of them (mir-182, mir-141 and mir-205) performing optimally. Conclusion: These results suggest the potential use of this non-invasive method of sampling for miRNA expression studies. Furthermore miRNA overexpression could serve as an ancillary or reflex test for optimal identification of malignant samples especially in morphologically inadequate samples. © Ivyspring International Publisher.

Background. Methylation of the human papillomavirus (HPV) DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within the L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV16 genotype. Methods. Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results. We found that the L1 HPV16 mean methylation (±SD) significantly increased with disease severity (cervical intraepithelial neoplasia [CIN] 3, 17.9% [±7.2] vs CIN2, 11.6% [±6.5], P < .001 or vs CIN1, 9.0% [±3.5], P < .001). Mean methylation was a good predictor of CIN3+ cases; the area under the curve was higher for sites 5611 in the prediction of CIN2+ and higher for position 7145 for CIN3+. The evaluation of different methylation thresholds for the prediction of CIN3+ showed that the optimal balance of sensitivity and specificity (75.7% and 77.5%, respectively) and positive and negative predictive values (74.7% and 78.5%, respectively) was achieved for a methylation of 14.0% with overall accuracy of 76.7%. Conclusions. Elevated methylation level is associated with increased disease severity and has good ability to discriminate HPV16-positive women that have high-grade disease or worse. © The Author 2017.

Background: γH2AX is a protein biomarker for double-stranded DNA breakage; its expression was studied in cervical squamous intraepithelial lesions and carcinomas. Methods: Immunostaining for phospho-γH2AX was performed in sections from histologically confirmed cervical SIL and carcinomas, as well as from normal cervices used as controls. In total, 275 cases were included in the study: 112 low grade SIL (LGSIL), 99 high grade SIL (HGSIL), 24 squamous cell carcinoma (SCC), 12 adenocarcinoma and 28 cervical specimens with no essential lesions. Correlation of histological grading, high risk vs. low risk HPV virus presence, activated vs. non-activated status (by high risk HPV mRNA expression) and γH2AX expression in both basal and surface segments of the squamous epithelium was performed. Results: Gradual increase of both basal and surface γH2AX expression was noted up from normal cervices to LGSIL harboring a low risk HPV type, to LGSIL harboring a high risk virus at a non-activated state (p

Background: Methylation of the HPV DNA has been proposed as a novel biomarker. Here, we correlated the mean methylation level of 12 CpG sites within L1 gene, to the histological grade of cervical precancer and cancer. We assessed whether HPV L1 gene methylation can predict the presence of high-grade disease at histology in women testing positive for HPV 16 genotype. Methods: Pyrosequencing was used for DNA methylation quantification and 145 women were recruited. Results: We found that the L1 HPV16 mean methylation (+/-SD) significantly increased with disease severity [CIN3=17.9%(±7.2) vs CIN2=11.6%(±6.5), p

Study Objective: To compare the prevalence of human papillomavirus (HPV) genital infection among prepubertal children, sexually active and not sexually active adolescents, and assess potential risk factors for transmission. Design: Prospective study. Setting: Outpatient adolescent health clinic. Participants: Ninety-five girls aged 2-21 years; 38 sexually active adolescents (group A), 28 not sexually active adolescents (group B), and 29 prepubertal children (group C). Interventions: Participants' vaginal or cervical specimens were tested for HPV with the CLART HPV 2 assay (Clinical Array Technology, Genomica, Madrid, Spain) and for cytological abnormalities with liquid-based cytology. Main Outcome Measures: Differences in prevalence of low- and high-risk HPV infections among the 3 groups. Results: Genital HPV was detected in 37.9% (36/95) of all participants; 47.4% (18/38) of group A, 28.6% (8/28) of group B, and 34.5% (10/29)of group C (P = .27). Multiple HPV infection was detected in 26.3% (10/38), 10.7% (3/28), and 13.8% (4/29) of groups A, B, and C, respectively (P = .21). High-risk genotypes were detected in 47.4% (18/38), 28.6% (8/28), and 24.1% (7/29) of groups A, B, and C, respectively (P = .10). Main high-risk genotypes were HPV 16 (27%, 10/37), HPV 31 (21.6%, 8/37 ), HPV 35 (13.5%, 5/37), HPV 53 (13.5%, 5/37), and low-risk HPV 6 (18.9%, 7/37). Sexual activity was associated with increased risk for genital high-risk HPV infection (odds ratio = 3.41; 95% confidence interval, 1.19-9.78); specifically with HPV 33 and HPV 51. Forty percent of sexually active adolescents with normal cervical cytology were infected with high-risk HPV types. Family history of skin HPV was positively associated with genital HPV in the sexually active group (odds ratio = 2.01; 95% confidence interval, 1.17-3.46). Conclusion: Timeline and target population for HPV vaccination might need to be reappraised, in view of significant nonsexual transmission of genital HPV so early in childhood. © 2015 North American Society for Pediatric and Adolescent Gynecology.

Purpose: Cytomegalovirus (CMV) reactivation, a significant cause of morbidity and mortality in immunosuppression, may affect "immunocompetent" seropositive critically ill patients. The aim of this prospective, observational study was to define the incidence, risk factors, and the association with morbidity and mortality of CMV reactivation in a general population of critically ill immunocompetent patients. We also studied the relationship between reactivation and patients' inflammatory response, as expressed by cytokine levels and stress up-regulation by salivary cortisol. Methods: This study included mechanically ventilated CMV-seropositive patients. A quantitative real-time polymerase chain reaction (PCR) was performed for CMV plasma DNAemia determination, upon intensive care unit (ICU) admission and weekly thereafter until day 28. Cytomegalovirus reactivation was defined as CMV plasma DNAemia greater than or equal to 500 copies/mL. Upon ICU admission, interferon γ, interleukin (IL) 10, IL-17A, IL-2, IL-6, and tumor necrosis factor α were quantified in plasma, and morning saliva was obtained to measure cortisol. Disease severity was assessed by Acute Physiology and Chronic Health Evaluation II score, whereas the degree of organ dysfunction was quantified by Sequential Organ Failure Assessment score. Mortality, duration of mechanical ventilation, and ICU length of stay were recorded. Results: During the study period, 80 (51 men) patients with a median age of 63 years fulfilled the inclusion criteria. Reactivation of CMV occurred in 11 patients (13.75%). Median day of reactivation was day 7 post ICU admission. Total number of red blood cell units transfused (odds ratio [OR], 1.50; confidence interval [CI], 1.06-2.13; P = .02) and C-reactive protein levels upon ICU admission (OR, 1.01; CI, 1.00-1.02; P = .02) were independently associated with CMV reactivation. High IL-10 was marginally related to reactivation (P = .06). Sequential Organ Failure Assessment scores were higher in the group with CMV reactivation compared with patients without reactivation during the entire 28-day observation period (P < .006). Salivary cortisol, mortality, length of ICU stay, and duration of mechanical ventilation were similar in the 2 groups. Conclusions: Cytomegalovirus reactivation occurred in 13.75% of critically ill, immunocompetent patients. The degree of inflammation and the total number of transfused red blood cells units constituted risk factors. Cytomegalovirus reactivation was associated with more severe of organ dysfunction, but not with a worse clinical outcome. © 2014 Elsevier Inc.