SummaryPurposeMolecular markers are only occasionally used in diagnostics of oral squamous cell carcinoma (OSCC), even though they could influence decision making in individually designed cancer therapies. We analyzed the predictive value of the markers HPV, p16, and HMGA2 and the TNM classification in regard to survival and recurrence rates.Material and MethodsA total of 91 OSCC cases were included in this study, with a follow up of up to 131 months. HPV-DNA was present in 7 carcinomas. p16 was detected by immunohistochemical staining in 14 samples. HMGA2 expression was determined by real-time quantitative polymerase chain reaction (qRT-PCR). Overexpression of HMGA2 was found to vary between 32-fold and 32,000-fold compared to nondysplastic tissue.ResultsCox regression analysis showed that age, sex, smoking status, use of alcohol, human papillomavirus (HPV), and tumor size had no significant effect on overall and progression-free survival. HMGA2 and N-status showed significant effects on overall (HMGA2: p = 0.049; N1: p = 0.019; N2: p = 0.02) and disease-free survival (HMGA2: p = 0.057; N1: p = 0.198; N2: p = 0.02). P16 appeared to be borderline significant but the χ2 indicated that p16 and N were correlated.ConclusionOur results suggest that HMGA2 expression may have the potential to allow a more precise prognosis on survival in patients with OSCC.

Of 34 breeds kept in the Czech Republic 45,604 sheep were genotyped for codons 136, 154 and 171 in the prion protein gene (PRNP) during the years 2006–2014. In this cohort, haplotypes ARR, ARQ, ARH, AHQ, VRQ, AHR and ARK were detected. The haplotype AF141RQ associated with susceptibility to atypical scrapie was observed in nine out of 30 breeds analysed for this purpose. In addition, six rare nonsynonymous substitutions producing haplotypes AT137RQ, AN138RQ, AG151RQ, AH151RQ, ARL168Q and ARQE175 were identified in various breeds. Due to their low frequencies, these polymorphisms are of no potential importance for the breeding programme. With regard to their genetic particularity, Sumavka, Valachian and Cameroon breeds were screened for additional polymorphisms. Further haplotypes, AR143RQ and AS146RQ, were found in Sumavka and Cameroon, and in Valachian sheep, respectively. Frequencies of the ARR (resistance-associated), VRQ (susceptibility-associated) haplotypes, and of the most resistant ARR/ARR genotype calculated for sheep born in the years 2001–2003 and 2011–2013 documented effects of the 10 year-lasting national breeding programme. The total frequency of ARR doubled from 36.8 to 75.8 %, while the frequency of VRQ decreased from 4 to 0.7 %. The total frequency of the ARR/ARR genotype increased from 17.7 to 59 %. These data show that the national scrapie resistance breeding programme has had an important desirable effect on haplotype and genotype frequencies of PRNP in Czech sheep.

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect three Encephalitozoon species in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked with Encephalitozoon intestinalis spores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 10² to 10⁴ spores/ml of feces, a value which represented a significant improvement over that achieved by staining (> or =1.0 x 10⁶ spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of three Encephalitozoon species (E. intestinalis , E. cuniculi , and E. hellem ). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detect Encephalitozoon species.

The objective of this study was to compare the MagNA Pure 96 Pathogen Universal protocol to the MagNA Pure LC DNA Isolation Kit III that is well established in our microbiological laboratory. For this we isolated nucleic acids from ten different sample materials: body fluids (Fluids, urine, sputum), feces, swabs, as well as whole blood, EDTA-, Citrate-Plasma and serum. This spectrum is typical for a microbiology laboratory and includes easy and difficult-to-process samples, as sputum and stool are. Into these sample types we spiked classified pathogen stocks from in-house cultures. Subsequently Real-time PCR analysis of fungal, bacterial and viral targets was done using the LightCycler^® 480 Instrument.The 200 μl protocol performed effi cient and sensitive nucleic acid extraction from up to 96 samples requiring less than 60 minutes runtime. It yielded superior or similar results compared to the respective MagNA Pure LC protocol. Higher input volumes, 500 or 1000 μl, available only for the MagNA Pure 96 System, generally increased sensitivity of detection. Direct use of some routine microbiological samples without dilution by pretreatment reagents, resulted in improved Crossing Point (CP) values. For normal sample types including whole blood this made pretreatment dispensable. A pretreatment step did however improve pathogen detection in the more diffi cult-to-process samples, such as sputum, swabs and stool. Our results demonstrate that both methods allow efficient and convenient isolation of high quality nucleic acid. Compared to the Viral NA Universal protocol, the Pathogen Universal protocol is further optimized for diverse sample materials, including difficult samples and for the detection of various pathogen types. The MagNA Pure 96 Instrument thus enables high throughput sample preparation applications in microbiology laboratories.