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 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.

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.