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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.

Human papillomavirus 16 (HPV16) infection is the aetiologic factor for the development of cervical dysplasia and is regarded as highly carcinogen, because it is implicated in more than 50% of cervical cancer cases, worldwide. The tumourigenic potential of HPV16 has triggered the extensive sequence analysis of viral genome in order to identify nucleotide variations and amino acid substitutions that influence viral oncogenicity and subsequently the initiation and progression of cervical cancer. Nowadays, specific mutations of HPV16 DNA have been associated with an increased risk of high-grade squamous intraepithelial lesions and invasive cervical cancer (ICC) development, including E6: Q14H, H78Y, L83V, Epsilon 7: N29S, S63F, E2: H35Q, P219S, T310K, E5: I65V, whereas highly conserved regions of viral DNA have been extensively characterised. In addition, numerous novel HPV16 mutations are observed among the studied populations from various geographic regions, hence advocating that different HPV16 strains seem to emerge with different tumourigenic capacities. The present review focuses on the variability of the early genes and the long control region, emphasising on the association of specific mutations with the development of severe dysplasia. Finally, it evaluates whether specific regions of HPV16 DNA are able to serve as valuable biomarkers for cervical cancer risk.

Breast cancer is the most common type of cancer in females and is the leading cause of cancer-associated death among women, worldwide. The present study aimed to measure the serum levels of fatty acid-binding protein 4 (FABP4), retinol binding protein 4 (RBP4) and the MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex in women diagnosed with breast cancer. Serum levels of the examined proteins were determined in the peripheral blood of patients via ELISA. Furthermore, whether the concentration of each protein was associated with breast cancer growth, molecular subtype, BMI, postmenopausal status, diabetes and the social background of patients was assessed. Women with invasive breast cancer demonstrated significantly higher levels of FABP4 (P=0.008). Additionally, considerably elevated FABP4 levels were demonstrated specifically in Luminal breast cancer cases (P

The increased prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has made essential the design of quicker tests for CPE detection. In the present study, a simple and rapid assay was developed based on measurement of the hydrolytic activity of imipenem at a final concentration of 65 µg/mL (100 µM) through ultraviolet–visible (UV-Vis) spectrophotometry. All measurements were conducted at 297 nm. A total of 83 carbapenem-non-susceptible CPE, consisting of Klebsiella pneumoniae clinical isolates and genotypically characterised as KPC-, VIM-, NDM- or OXA-48-producers, were tested. For comparison, 30 carbapenem-non-susceptible clinical isolates, consisting of Escherichia coli and K. pneumoniae and genotypically confirmed as non-CPE, were also examined. The spectrophotometric assay enabled efficient discrimination of CPE from non-CPE isolates even in 45 min (P < 0.0001). Moreover, the presence of phenylboronic acid (PBA) or ethylene diamine tetra-acetic acid (EDTA) in the reaction mixture was able to inhibit the hydrolytic capacity of KPC- or metallo-β-lactamase (MBL)-producers, respectively, while the hydrolytic activity of OXA-48-producing strains was not affected by the presence of these inhibitors (P < 0.001). The newly developed assay presented 100% sensitivity and specificity to detect and differentiate KPC-, MBL- and OXA-48-producers compared with genotypic characterisation. Thus, the proposed spectrophotometric method can be considered as an easy, fast, accurate and cost-effective diagnostic tool for screening carbapenem-non-susceptible K. pneumoniae isolates in the clinical laboratory. © 2020 Elsevier Ltd and International Society of Antimicrobial Chemotherapy

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.

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

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.

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.

Purpose. Polymorphic variability in the tumour-suppressor protein p53 at codon 72 has a considerable impact on cervical cancer development. The present study clarified the association between p53 codon 72 genotypes and the risk of cervical disease in Greek patients. We also examined whether the presence of specific p53 genotypes in combination with HPV16 variants or E6 T350G sequence variation can modify an individual’s susceptibility to cervical disease. Methodology. The analysis of p53 genotypes was performed through PCR-RFLP. Sequence and phylogenetic tree analyses of the HPV16 E6 gene were also performed in order to identify HPV16 variants and T350G sequence variation. Results/Key findings. The outcomes of the present analysis revealed that women who are homozygous for the arg genotype are at a 4.17-fold higher risk of developing HPV16-associated HSIL+ (OR=4.17, 95%CI:1.48-4.9, P=0.0049). Moreover, p53 arg/arg patients infected by an HPV16 prototype strain were associated with an increased risk of more severe lesions, while a significant relationship between the p53 arg/arg genotype in patients with T350G sequence variation and the risk of highgrade squamous intraepithelial lesions (HSILs) was revealed. Conclusion. The oncogenic potential of the virus is increased by the presence of the p53 arg/arg genotype in the Greek population in such a way that the specific protein interaction E6 (L83V)-p53 (Arg-72) can modify an individual’s susceptibility to cervical disease. © 2017 The Authors.