Analytical and clinical evaluation of the HPV DNA Array E1-based genotyping assay

Background: HPV infection has shown to be mandatory for development of cervical dysplasia. Consequently, molecular HPV detection is used for cervical cancer screening, especially for genotype-specific persistence. Aim of this study was to evaluate the analytical and clinical performance of HPV DNA Array, an E1-based genotyping test for identification of 29 HPV types: 6,11,16,18,26,31,33,35,39,40,42,44,45,51,52,53,54,56, 58,59,66,67,68,69,70,73,82,85 and 97. Methods: HPV DNA Array is based on a multiplexed PCR followed by reverse hybridization in a 96-well format with automated visual readout, capable of high-throughput in a time-effective manner. Technical performance of the assay was assessed with cervical cancer cell lines with known HPV status, and preselected clinical cervical scrapings genotyped by multiplexed genotyping (MPG) with Luminex readout. Intra- and inter-laboratory reproducibility experiments were performed to ensure reliability of the assay. The clinical evaluation was performed against the reference assays, BS-GP5+/6+ MPG-Luminex, with 600 cervical smear samples of a referral population, and the FDA-approved Cobas 4800 HPV test on a study population of 500 cervical samples. Results: HPV DNA Array identified the intrinsic HPV genotype in all cervical cancer cell lines and demonstrated a high sensitivity for the HPV16 probe (1 cell/PCR reaction), HPV18 and 45 probes (100 cells/PCR reaction). When compared with MPG within the analytical study, HPV DNA Array showed good agreement of 92.2% for HPV detection irrespective of type (κ=0.601), and demonstrated high agreement for HPV16 (80.7%, κ=0.836), and HPV18 (86.7%, κ=0.925). Furthermore, high intra-/inter-laboratory reproducibility was observed (90.9%-100%). HPV DNA Array detected CIN2+ lesions with a sensitivity of 90.2%, identical to that of MPG-Luminex. Sensitivity for detection of CIN3+ lesions was 90.3%, as compared with 88.7% of MPG-Luminex. HPV DNA Array demonstrated very good agreement for HPV detection, irrespective of type, of 91.5% (κ=0.832) within the clinical evaluation study. HPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+/CIN3+ detection same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+, than cytology (100% vs. 13.6%). The agreement with Cobas 4800 for HPV detection, irrespective of type, was 81.4% (κ=0.613). The agreement for HPV16 was 92.8% (κ=0.929), and for HPV18 54.2% (κ=0.681). Conclusion: HPV DNA Array has demonstrated a good performance in HPV and CIN2+ detection with high reproducibility and it is capable of extended HPV genotyping by a technically simple method. HPV DNA Array could be considered for extended HPV genotyping of cervical smears and in organized screening programs and potentially in low resource settings.Hintergrund: Eine HPV-Infektion ist obligatorisch für die Entwicklung von zervikalen Dysplasien. Dabei wird der molekulare HPV-Nachweis zur Früherkennung von Gebärmutterhalskrebs und genotypspezifischer Persistenz eingesetzt, insbesondere für letztere. Ziel dieser Studie war es, die analytische und klinische Leistungsfähigkeit des HPV-DNA-Arrays, eines E1-basierten multiplexen PCR-Tests zur Identifizierung von 29 HPV-Typen: 6,11,16,18,26,31,33,35,39,40,42,44,45,51,52,53,54,56,58,59,66,67,68,69, 70,73,82,85 und 97, zu bewerten. Methoden: HPV DNA Array basiert auf einer Multiplex-PCR mit anschließender Rück-Hybridisierung im 96-Well-Format mit automatischer visueller Auslesung, die einen hohen Durchsatz in zeitsparender Weise ermöglicht. Die technische Leistung des Arrays wurde mit Zervixkarzinom-Zelllinien mit bekanntem HPV-Status und vorselektierten klinischen Zervixabstrichen, die durch Multiplex-Genotypisierung (MPG) mit Luminex-Auslesung genotypisiert wurden, bewertet. Intra- und interlaboratorische Reproduzierbarkeit wurde durchgeführt, um die Zuverlässigkeit des Arrays zu gewährleisten. Die klinische Auswertung erfolgte gegenüber den Referenz-Assays BS-GP5+/6+ MPG-Luminex mit 600 Zervixabstrichen von zur Abklärung überwiesenen Patienten und Cobas 4800 HPV-Test an einer Studienpopulation von 500 Zervixproben. Ergebnisse: Das HPV DNA Array identifizierte den intrinsischen HPV-Genotyp in allen zervikalen Krebszelllinien und zeigte eine hohe Sensitivität für HPV16 Sonden (1 Zelle/PCR-Reaktion) sowie HPV18 und 45 Sonden (100 Zellen/PCR-Reaktion). Im Vergleich zu MPG in der analytischen Studie zeigte HPV DNA Array in der analytischen Studie eine gute Übereinstimmung von 92,2% für den HPV-Nachweis unabhängig vom Typ (κ=0,601) und eine hohe Übereinstimmung für HPV16 (80,7%, κ=0,836) und HPV18 (86,7%, κ=0,925). Darüber hinaus wurde eine hohe intra- bzw. interlaboratorische Reproduzierbarkeit beobachtet (90,9%-100%). HPV DNA Array detektierte CIN2+ Läsionen mit einer Sensitivität von 90,2%, identisch mit der von MPG-Luminex. Der Nachweis von CIN3+ Läsionen erfolgte mit einer Sensitivität von 90,3%, verglichen mit 88,7% bei MPG-Luminex. Es zeigte sich eine sehr gute Übereinstimmung für den HPV-Nachweis, unabhängig vom Typ, von 91,5% (κ=0,832) innerhalb der klinischen Evaluationsstudie. HPV DNA Array zeigte eine sehr hohe Sensitivität von 100% für den CIN2+/CIN3+ Nachweis so wie der Cobas 4800. HPV DNA Array zeigte eine höhere Sensitivität für CIN2+, als die Zytologie (100% vs. 13.6%). Die Übereinstimmung mit Cobas 4800 zur HPV-Erkennung, unabhängig vom Typ, betrug 81,4% (κ=0,613). Die Übereinstimmung für HPV16 betrug 92,8% (κ=0,929) und für HPV18 54,2% (κ=0,681). Schlussfolgerung: HPV DNA Array hat eine gute Zuverlässigkeit bei der HPV- und CIN-Detektion mit hoher Reproduzierbarkeit gezeigt und ist in der Lage, die HPV-Genotypisierung durch eine technisch einfache Methode zu erweitern. HPV DNA Array könnte für die erweiterte HPV-Genotypisierung von Zervixabstrichen und in organisierten Screening-Programmen sowie potentiell bei geringer Ressourcenverfügbarkeit in Betracht gezogen werden

CIN2+ detection of the HPV DNA Array genotyping assay in comparison with the Cobas 4800 HPV test and cytology

BACKGROUND: HPV DNA Array is an E1-targeting PCR genotyping test, with capability of distinguishing 18 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and 11 low-risk HPV types (6, 11, 40, 42, 44, 54, 67, 69, 70, 85, 97). HPV DNA Array uses multiplex PCR for E1-gene sequence amplification. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID ELISPOT reader. METHODS: Aim of the study was to evaluate the clinical performance of the assay against internationally accepted and FDA approved Cobas 4800 HPV test (Roche Diagnostics). Study population comprised of 500 cervical samples. RESULTS: HPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+ detection than cytology (100% vs. 13.6%). The agreement to Cobas 4800 for HPV detection, irrespective of type, was 81.4% with κ = 0.613. The agreement for HPV 16 was 92.8% (κ = 0.929), and for HPV 18 54.2% (κ = 0.681). CONCLUSION: HPV DNA Array demonstrated good clinical performance for detection of high-grade lesions, and may be considered for usage in a screening setting

Clinical performance of the HPV DNA Array genotyping assay in detection of CIN2+ lesions with BS GP5+/6+ MPG Luminex tested cervical samples

Human papillomavirus (HPV) detection is used for screening of cervical cancer and genotype-specific persistence has shown to be mandatory for dysplasia development. Aim of this study was to evaluate the clinical performance of HPV DNA Array for cervical intraepithelial neoplasia 2+ (CIN2+) lesion detection. HPV DNA Array is a polymerase chain reaction-based assay that targets E1 sequences of 29 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and 97). The clinical evaluation was performed against the reference assay, BS-GP5+/6+ multiplex genotyping (MPG)-Luminex, with 600 cervical smear samples of a referral population. HPV DNA Array detected CIN2+ lesions with a sensitivity of 90.2%, identical to that of MPG-Luminex. Detection of CIN3+ lesions was with a sensitivity of 90.3%, as compared with 88.7% of MPG-Luminex. It demonstrated very good agreement for HPV detection, irrespective of type, of 91.5% (kappa = 0.832). HPV DNA Array is a simple and robust assay, with a short protocol of 4 hours hands-on time and automated readout by ELISpot AiDot software. It permits testing of up to 96 samples in one run and may be considered for use in organized screening programs and low resource settings

Comparative toxicity evaluation of targeted anticancer therapeutics in embryonic zebrafish and sea urchin models

Cancer drug resistance and poor selectivity towards cancer cells demand the constant search for new therapeutics. PI3K-Akt-mTOR and RAS-MAPK-ERK signaling pathways are key mechanisms involved in cell survival, proliferation, differentiation, and metabolism and their deregulation in cancer can promote development of therapy resistance. We investigated the effects of targeted inhibitors (wortmannin, GSK690693, AZD2014 and tipifarnib) towards these two pathways on early zebrafish and sea urchin development to assess their toxicity in normal, fast proliferating cells. PI3K inhibitor wortmannin and RAS inhibitor tipifarnib displayed highest toxicity while GSK690693, a pan-Akt kinase inhibitor, exhibited a less significant impact on embryo survival and development. Moreover, inhibition of the upstream part of the PI3K-Akt-mTOR pathway (wortmannin/GSK690693 co-treatment) produced a synergistic effect and impacted zebrafish embryo survival and development at much lower concentrations. Dual mTORC1/mTORC2 inhibitor AZD2014 showed no considerable effects on embryonic cells of zebrafish in concentrations substantially toxic in cancer cells. AZD2014 also caused the least prominent effects on sea urchin embryo development compared to other inhibitors. Significant toxicity of AZD2014 in human cancer cells, its capacity to sensitize resistant cancers, lower antiproliferative activity against human normal cell lines and fast proliferating embryonic cells could make this agent a promising candidate for anticancer therapy

Characterization of Human Papillomavirus prevalence and risk factors to guide cervical cancer screening in the North Tongu District, Ghana

Introduction: This population-based study aimed to fill the knowledge gap on Human Papillomavirus (HPV) prevalence and associated sociodemographic risk factors of the general population in the North Tongu District, Ghana. These results are needed to guide cervical cancer prevention efforts, as the leading type of female cancers. Methods: A cross-sectional study including 2002 women in the North Tongu District, Ghana investigated HPV prevalence and associated sociodemographic risk factors. Women were recruited by geographical distribution through the local community-based health system and samples collected using a self-sampling device. For HPV genotyping BSGP5+/6+-PCR with Luminex-MPG readout was used. Multivariate logistic regression analyzed sociodemographic risk factors for HPV positivity. Results: Of 2002 self-collected samples, 1943 were eligible, contained sufficient DNA and provided valid HPV genotyping results. Prevalence of single high risk HPV types was 32.3% and of multiple high risk types 9.7%. The five most common detected HPV types were HPV16 (7.4%; 95%CI: 6.3–8.7), HPV52 (7.2%; 95%CI: 6.1–8.5), HPV35 (4.8%; 95%CI: 3.9–5.8), HPV59 (4.7%; 95%CI: 3.8–5.8), HPV56 (3.9%; 95%CI: 3.1–4.8). Highest prevalence was observed among women aged 18–24 years, while age 25–54 years was inversely associated with high risk HPV positivity in multivariate analysis. Sociodemographic risk factors identified were i) having any sexual partner, ii) more partners increased the odds for high risk HPV positivity, iii) independently from this marital status, in particular not being married. Discussion & conclusion: Most importantly, the high risk HPV prevalence detected from this study is higher than estimates reported for Western Africa. This needs be considered, when deciding on the cervical cancer screening algorithms introduced on a wider scale. Follow-up and triage, depending on the methods chosen, can easily overburden the health system. Self-sampling worked well and provided adequate samples for HPV-based screening. Women with increasing number of sexual partners and not being married were found to have higher odds of being high risk HPV positive, therefore could be a higher prioritized screening target group

Stress i förskolan : Små barn i stora barngrupper

This study aimed to investigate whether young children between the ages of 1-3 experience stress as a result of larger class sizes and the manner in which to mitigate this stress. I have also investigated whether educators may induce stress in children and generally whether stress amongst adults is transferrable to children. I propose the following questions: How do educators perceive stress in children's groups? How do children react to stress? How to prevent stress in preschool children? This study is based on a qualitative method with semi-structured interviews and participant observations. My theoretical approach to this study is the sociocultural perspective. The literature points to the fact that various stress-related situations and unstable relationships in preschool can contribute to the stress levels of children. Interviews and observational studies have found that there is a positive relationship between classroom size and stress among children. Classroom sizes lead to stressed teachers, less time devoted to each individual child, loud noises in the children's groups, fewer staff as well as less space for children to play and move around in. These are only some of the burdens of larger classroom sizes. In addition, the investigation showed that for children who do not speak, upon interaction they tended to mimic their educator’s stressful behaviour and thus felt stressed themselves. The educator’s stressful behaviour very strongly influenced the stress levels of all of the children in the group. Teachers had strategies to prevent and manage stress in kindergarten but also suggested organizational changes to divide the children more frequently into small groups, to adapt the physical environment to accommodate for a larger group of children and to give children more time in the dining room

A DNA barcode library for the water mites of Montenegro

Water mites (Acari, Hydrachnidia) are a significant component of freshwater ecosystems inhabiting a wide range of aquatic habitats. This study provides a first comprehensive DNA barcode library for the water mites of Montenegro. DNA barcodes were analysed from 233 specimens of water mites morphologically assigned to 86 species from 28 genera and 15 families. In the course of the study, four species, i.e. Lebertia reticulata (Koenike, 1919), Atractides inflatipalpis K.Viets, 1950, A. latipes (Szalay, 1935) and Parabrachypoda montii (Maglio, 1924) were molecularly confirmed as new for Montenegro and three species, i.e. Protzia octopora Lundblad, 1954, Piona laminata (Thor, 1901) and Unionicola ypsilophora (Bonz, 1783) are new for the Balkan Peninsula. Results are analysed using the Barcode Index Number system (BIN) and the Refined Single Linkage (RESL) of BOLD. The BIN assigned sequences to 98 clusters, while the RESL reveal 103 operational taxonomic units (OTUs). Unique BINs were revealed for 72 species (83.7%), whereas twelve species (14%) were characterised by two BINs and two species (2.3%) with three BINs. Amongst the studied taxa, 14 species were found with a high intraspecific sequence divergences (˃ 2.2%), emphasising the need for additional comprehensive morphological and molecu­lar analysis of these species

Adverse plasma fatty acid composition in patients with femoral neck fracture

Our study was aimed to examine the status of plasma fatty acids (FAs), inflammatory markers and lipid peroxidation in patients with femoral neck fractures. Study included 20 patients (64-86 years) with femoral neck fractures, indicated for surgery, and control group of 17 elderly subjects, without fractures or serious chronic diseases. Plasma was obtained during the first 12 hours postfracture and presurgery, and 7 days postop. Compared to control, patients had significantly higher saturated FA (SFA) and monounsaturated FA, as well as increased TNF-α and IL-6. In opposite, levels of individual and total n-6 polyunsaturated FA (PUFA), individual and total n-3 PUFA, n-6/n-3 ratio and levels of thiobarbituric acid reactive substances (TBARS) were markedly lower in the patient than controls. On the 7th day after the surgery we showed a further rise in the SFA, oleic acid and TNF-α, and reductions of n-6 PUFA and IL-6. Taken together our results suggest that altered FA status, especially reduced PUFA, may influence hip fracture repair and even contribute to femoral fractures susceptibility in the elderly. Potential benefit from nutritional intervention with PUFA in prevention and/or fracture healing should be considered.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Potential application of low molecular weight excipients for amorphization and dissolution enhancement of carvedilol

In this study, four low molecular weight (LMW) excipients, tryptophan (TRY), phenylalanine (PHE), lysine (LYS) and saccharin (SAC) were evaluated as co-formers to generate co-amorphous systems (CAMS) by ball milling with carvedilol (CRV). Mixtures of CRV and LMW excipient in 1:0.5, 1:1 and 1:2 drug:excipient molar ratios were ball milled and analysed by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), Fourier transform (FT-IR) infrared spectroscopy and dissolution testing. CAMS were formed by milling of a mixture of CRV with TRY in 1:2 M ratio and SAC in 1:1 M ratio, while amorphization of only CRV was achieved in other mixtures with SAC. In other samples containing TRY and PHE, milling resulted in partial amorphization, while LYS was the least suitable excipient for the amorphization of CRV. Unexpectedly, the highest supersaturation of CRV was achieved from samples containing CRV and LYS in 1:1 and 1:2 M ratios, despite the absence of a significant reduction in CRV crystallinity upon milling of these samples. Increase of hydrophobic surface area caused by milling of samples with TRY and PHE and agglomeration during dissolution testing of samples containing SAC are likely causes of poor dissolution performance of mixtures containing fully or partially amorphous CRV