Adapting Rapid Diagnostic Tests to Detect Historical Dengue Virus Infections.

The only licensed dengue vaccine, Dengvaxia®, increases risk of severe dengue when given to individuals without prior dengue virus (DENV) infection but is protective against future disease in those with prior DENV immunity. The World Health Organization has recommended using rapid diagnostic tests (RDT) to determine history of prior DENV infection and suitability for vaccination. Dengue experts recommend that these assays be highly specific (≥98%) to avoid erroneously vaccinating individuals without prior DENV infection, as well as be sensitive enough (≥95%) to detect individuals with a single prior DENV infection. We evaluated one existing and two newly developed anti-flavivirus RDTs using samples collected >6 months post-infection from individuals in non-endemic and DENV and ZIKV endemic areas. We first evaluated the IgG component of the SD BIOLINE Dengue IgG/IgM RDT, which was developed to assist in confirming acute/recent DENV infections (n=93 samples). When evaluated following the manufacturer's instructions, the SD BIOLINE Dengue RDT had 100% specificity for both non-endemic and endemic samples but low sensitivity for detecting DENV seropositivity (0% non-endemic, 41% endemic). Sensitivity increased (53% non-endemic, 98% endemic) when tests were allowed to run beyond manufacturer recommendations (0.5 up to 3 hours), but specificity decreased in endemic samples (36%). When tests were evaluated using a quantitative reader, optimal specificity could be achieved (≥98%) while still retaining sensitivity at earlier timepoints in non-endemic (44-88%) and endemic samples (31-55%). We next evaluated novel dengue and Zika RDTs developed by Excivion to detect prior DENV or ZIKV infections and reduce cross-flavivirus reactivity (n=207 samples). When evaluated visually, the Excivion Dengue RDT had sensitivity and specificity values of 79%, but when evaluated with a quantitative reader, optimal specificity could be achieved (≥98%) while still maintaining moderate sensitivity (48-75%). The Excivion Zika RDT had high specificity (>98%) and sensitivity (>93%) when evaluated quantitatively, suggesting it may be used alongside dengue RDTs to minimize misclassification due to cross-reactivity. Our findings demonstrate the potential of RDTs to be used for dengue pre-vaccination screening to reduce vaccine-induced priming for severe dengue and show how assay design adaptations as well quantitative evaluation can further improve RDTs for this purpose.

Novice learner in-plane ultrasound imaging: which visualization technique?

BACKGROUND AND OBJECTIVES: Needle guidance under ultrasound is an acquired skill requiring fine motor control. Maintaining the image of an advancing needle in the plane of an ultrasound beam may be performed with the probe and needle orientated along the visual axis (AL) or across the visual axis (AC). This study was undertaken to determine if orientation affected task performance. METHODS: Twenty-four relative novices were tasked to perform guided punctures to a target in a pork phantom using each technique 5 times. The technique first used was randomly chosen from a sealed envelope. The time taken to guide the needle to target and the accuracy of needle imaging were recorded. RESULTS: The mean time to locate the target was significantly faster for the AL technique, compared with the AC technique (group AL, 35.7, vs group AC, 58.6 seconds; P < 0.0001, Wilcoxon matched-pairs signed rank test). The mean imaging quality score was also significantly better when needle advancement was along the visual axis (group AL, 1.37, vs group AC, 1.64; P = 0.05). CONCLUSIONS: Advancing the needle along the visual axis was associated with improved task completion speed and quality of needle imaging. This ergonomic pattern, therefore, may be the more appropriate choice for novices learning ultrasound-guided in-plane needle imaging.

Protein folding in Escherichia coli: the chaperonin GroE and its substrates.

A brief summary of the role of DnaK and GroE chaperones in protein folding precedes a discussion of the role of GroE in Escherichia coli. We consider its obligate substrates, the 8 that are both obligate and essential, and the prospects for constructing a mutant that could survive without it. Structural features of GroE-dependent polypeptides are also considered.

Prion protein accumulation and neuroprotection in hypoxic brain damage.

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

Male infertility and DNA damage in Doppel knockout and prion protein/Doppel double-knockout mice.

The prion protein (PrP) and Doppel (Dpl) have many structural and biochemical properties in common, leading to the suggestion that the lack of an obvious phenotype in PrP-deficient mice maybe because of compensation by Dpl. To test this hypothesis and also investigate the function of Dpl we have generated Prnd(-/-) and Prnp(-/-)/Prnd(-/-) mouse lines. Both develop normally and display an identical male sterility phenotype that differs from that reported for another Prnd(-/-) mouse line. Sperm from both our mutant lines were present at normal concentrations, had normal motility, and no morphological abnormalities. Despite only rarely fertilizing oocytes in vivo, because of an inability to perform the acrosome reaction, mutant sperm were capable of fertilization in vitro, albeit at reduced rates compared to wild type. Elevated levels of oxidative DNA damage were found in both types of mutant sperm and resulting embryos failed at an early stage. Therefore we found no evidence that Dpl compensates for the loss of PrP function in mutant mouse lines, but it does have an important anti-oxidant function necessary for sperm integrity and male fertility.

Prion protein accumulation in eyes of patients with sporadic and variant Creutzfeldt-Jakob disease.

PURPOSE: Creutzfeldt-Jakob disease (CJD) primarily affects the brain. This study was conducted to assess the possible involvement of the eye in sporadic and variant CJD by testing for the presence of the disease-associated, protease-resistant isoform of the prion protein (PrP(Sc)) in ocular tissue. METHODS: Human eyes from donors with CJD and non-prion neurodegenerative disease control eyes were studied. In situ hybridization and Western blot analysis were used to determine the normal pattern of cellular prion protein (PrP(C)) expression. Western blot analysis and immunohistochemistry were then used to determine the localization, abundance, and isotype of PrP(Sc) in eyes in CJD. RESULTS: PrP(C) was expressed in the nuclear layers of the retina. In both the sporadic and variant forms of CJD, PrP(Sc) accumulated throughout the synaptic layers of the retina. The levels of PrP(Sc) found in the retina were comparable with those found in the brain. Lower levels of PrP(Sc) could be found in the optic nerve, but no PrP(Sc) was detectable in other ocular tissues. The glycoform ratio of PrP(Sc) in the retina did not correspond to that found in the brain. CONCLUSIONS: Presumptive centrifugal spread of PrP(Sc) from the brain through the optic nerve occurs in two major types of CJD. PrP(Sc) is a marker of CJD infectivity. Given that routine decontamination may not remove PrP(Sc) from surgical instruments, a careful risk assessment should be made of possible iatrogenic spread of sporadic and variant CJD after surgery to the retina or optic nerve.

Properties of the cellular prion protein expressed in Xenopus oocytes.

The cellular prion protein (PrPC) from different species can be reproducibly expressed in Xenopus oocytes following injection of in vitro transcribed mRNAs. The level of PrPC accumulation increases with the amount of RNA injected and with the duration of incubation. PrPC expressed in oocytes is similar in size and abundance to PrPC protein in mouse brain and >100 ng of PrPC is expressed per oocyte allowing complete experiments to be carried out in single living cells. The protein is glycosylated, fully protease sensitive and expressed on the cell surface. Xenopus oocytes therefore provide a useful model system for the study of prion proteins and their associated disease processes.