Principles and analytical performance of Papilloplex® HR-HPV, a new commercial CE-IVD molecular diagnostic test for the detection of high-risk HPV genotypes.

The accurate detection and genotyping of high-risk human papillomavirus (HR-HPV) are critical for cervical cancer screening and epidemiological investigations. GeneFirst Papilloplex® HR-HPV is a new CE-IVD-marked real-time PCR test based on patented multiplex probe amplification technology. Papilloplex® HR-HPV provides the simultaneous detection and differentiation of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68a/b) in a single closed-tube reaction ensuring rapid, cost-effective, and contamination-free results. In this study, the analytical performance characteristics in terms of the assay's sensitivity, specificity, range, reproducibility, and cross-reactivity were evaluated. Papilloplex® HR-HPV provided sensitive detection and differentiation of 14 HR-HPV types with highly reproducible results. The differential HR-HPV specificity and sensitivity were further confirmed through the participation in the WHO HPV Laboratory Network Proficiency Study (2014). Overall, GeneFirst Papilloplex® HR-HPV assay demonstrated a robust analytical performance with reproducible and reliable results in the detection of HR-HPV genotypes.

Studies of mitochondrial and nonmitochondrial sources implicate nicotinamide adenine dinucleotide phosphate oxidase(s) in the increased skeletal muscle superoxide generation that occurs during contractile activity.

AIMS: The sources of cytosolic superoxide in skeletal muscle have not been defined. This study examined the subcellular sites that contribute to cytosolic superoxide in mature single muscle fibers at rest and during contractile activity. RESULTS: Isolated fibers from mouse flexor digitorum brevis loaded with superoxide and nitric-oxide-sensitive fluorescent probes, specific pathway inhibitors and immunolocalization techniques were used to identify subcellular sites contributing to cytosolic superoxide. Treatment with the electron transport chain complex III inhibitor, antimycin A, but not the complex I inhibitor, rotenone, caused increased cytosolic superoxide through release from the mitochondrial intermembrane space via voltage-dependent anion or Bax channels, but inhibition of these channels did not affect contraction-induced increases in cytosolic superoxide. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors decreased cytosolic superoxide at rest and following contractions. Protein and mRNA expression of NADPH oxidase subunits was demonstrated in single fibers. NOX2, NOX4, and p22(phox) subunits localized to the sarcolemma and transverse tubules; NOX4 was additionally expressed in mitochondria. Regulatory p40(phox) and p67(phox) proteins were found in the cytoplasm of resting fibers, but following contractions, p40(phox) appeared to translocate to the sarcolemma. INNOVATION: Superoxide and other reactive oxygen species generated by skeletal muscle are important regulators of muscle force production and adaptations to contractions. This study has defined the relative contribution of mitochondrial and cytosolic sources of superoxide within the cytosol of single muscle fibers at rest and during contractions. CONCLUSION: Muscle mitochondria do not modulate cytosolic superoxide in skeletal muscle but NADPH oxidase is a major contributor both at rest and during contractions.