Detection of Influenza type A virus by LightCycler RT-PCR

Using the Roche LightCycler we developed a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the Influenza A LightCycler RT-PCR (FA-LC-RTPCR) for the rapid detection of Influenza A. The assay was used to examine 178 nasopharyngeal aspirate (NPA) samples, from patients with clinically recognised respiratory tract infection, for the presence of Influenza A RNA. The results were then compared to a testing algorithm combining direct immunofluorescent assy (DFA) and a culture augmented DFA (CA-DFA) assay. In total, 76 (43%) specimens were positive and 98 (55%) specimens were negative by both the FA-LC-RTPCR and the DFA and CA-DFA algorithm. In addition, the FA-LC-RTPCR detected a further 4 (2%) positive specimens, which were confirmed by a conventional RT-PCR method. The high level of sensitivity and specificity, combined with the rapid turnaround time for results, makes the LC-RT-PCR assay suitable for the detection of Influenza A in clinical specimens.

Molecular assays for detection of human metapneumovirus

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

Crack detection in hollow section structures through coupled response measurements

Detection of a circumferential crack in a hollow section beam is investigated using coupled response measurements. The crack section is represented by a local flexibility matrix connecting two undamaged beam segments. This matrix defines the relationship between the displacements and forces across the crack section and is derived by applying fundamental fracture mechanics theory. The suitability of the mode coupling methodology is first demonstrated analytically. Laboratory test results are then presented for circular hollow section beams with artificially generated cracks of varying severity. It is shown that this method has the potential as a damage detection tool for mechanical structures. (C) 2002 Elsevier Science Ltd. All rights reserved.

Skin-friction measurements in high-enthalpy hypersonic boundary layers

Skin-friction measurements are reported for high-enthalpy and high-Mach-number laminar, transitional and turbulent boundary layers. The measurements were performed in a free-piston shock tunnel with air-flow Mach number, stagnation enthalpy and Reynolds numbers in the ranges of 4.4-6.7, 3-13 MJ kg(-1) and 0.16 x 10(6)-21 x 10(6), respectively. Wall temperatures were near 300 K and this resulted in ratios of wall enthalpy to flow-stagnation enthalpy in the range of 0.1-0.02. The experiments were performed using rectangular ducts. The measurements were accomplished using a new skin-friction gauge that was developed for impulse facility testing. The gauge was an acceleration compensated piezoelectric transducer and had a lowest natural frequency near 40 kHz. Turbulent skin-friction levels were measured to within a typical uncertainty of +/-7%. The systematic uncertainty in measured skin-friction coefficient was high for the tested laminar conditions; however, to within experimental uncertainty, the skin-friction and heat-transfer measurements were in agreement with the laminar theory of van Driest (1952). For predicting turbulent skin-friction coefficient, it was established that, for the range of Mach numbers and Reynolds numbers of the experiments, with cold walls and boundary layers approaching the turbulent equilibrium state, the Spalding & Chi (1964) method was the most suitable of the theories tested. It was also established that if the heat transfer rate to the wall is to be predicted, then the Spalding & Chi (1964) method should be used in conjunction with a Reynolds analogy factor near unity. If more accurate results are required, then an experimentally observed relationship between the Reynolds analogy factor and the skin-friction coefficient may be applied.

Preliminary comparison of three lightcycler pcr assays for the detection of herpes simplex virus in swab specimens

Three Herpes Simplex Virus LightCycler polymerase chain reaction assays were compared for the detection of herpes simplex virus in 48 swab specimens. The assays comprised of one in-house assay and two commercial kits: the Artus HSV LC RealArt PCR kit and the Roche LightCycler HSV 1/2 Detection kit. On the whole, the three assays had comparable sensitivities. However, differentiation of herpes simplex virus types 1 and 2 by melting curve analysis was problematic in all assays. Overall, the results highlight the limitations of typing herpes simplex virus by melting curve analysis.

The role of melanocortin-1 receptor polymorphism in skin cancer risk phenotypes

We have examined melanocortin-1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32-140) or weak r (OR = 5; 95% CI 3-11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild-type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.

Induction of metallothionein in human skin by routine exposure to sunlight: Evidence for a systemic response and enhanced induction at certain body sites

Expression of metallothionein, an antioxidant induced by a variety of stimuli including ultraviolet light, was quantitated by immunohistochemistry in the skin of males aged over 50 who had known short- and long-term exposures to sunlight. Skin punch biopsies were taken from two sites in each subject: the hand in all subjects and a range of other sites matched to patients with a previously excised primary melanoma. Metallothionein expression (strongest in the basal layers of the epidermis and primarily nuclear) was associated with both short- and long-term exposure to sunlight. A plateau of staining intensity was reached after 3 h sun exposure, within the previous 3 d before biopsy. Expression was also elevated in the nonexposed skin sites of subjects who had recent sun exposure, indicating a systemic response to exposure of remote sites. Using the skin of the hand to normalize responses to chronic exposure between individuals, the systemically modulated response to sunlight was significantly greater on the unexposed back than on other sites. The possibility of ultraviolet-induced cytokines selectively modifying the response of skin on a site-specific basis was investigated. The circulating leukocytes, but not lymphocytes, of two individuals exposed to 1 minimal erythema dose whole-body solar-simulated ultraviolet showed increased interleukin-6 mRNA 4 h after exposure. Interleukin-6 was not directly induced in these cell populations 4 h after ultraviolet A or ultraviolet B irradiation ex vivo . Leukocytes may therefore contribute to and amplify the systemic effects of ultraviolet-induced interleukin-6 and metallothionein expression.

Detection of West Nile Virus Infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in-New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.

Determination of the effect of lipophilicity on the in vitro permeability and tissue reservoir characteristics of topically applied solutes in human skin layers

In order to establish the relationship between solute lipophilicity and skin penetration (including flux and concentration behavior), we examined the in vitro penetration and membrane concentration of a series of homologous alcohols (C2-C10) applied topically in aqueous solutions to human epidermal, full-thickness, and dermal membranes. The partitioning/distribution of each alcohol between the donor solution, stratum corneum, viable epidermis, dermis, and receptor phase compartments was determined during the penetration process and separately to isolated samples of each tissue type. Maximum flux and permeability coefficients are compared for each membrane and estimates of alcohol diffusivity are made based on flux/concentration data and also the related tissue resistance (the reciprocal of permeability coefficient) for each membrane type. The permeability coefficient increased with increasing lipophilicity to alcohol C8 (octanol) with no further increase for C10 (decanol). Log vehicle:stratum corneum partition coefficients were related to logP , and the concentration of alcohols in each of the tissue layers appeared to increase with lipophilicity. No difference was measured in the diffusivity of smaller more polar alcohols in the three membranes; however, the larger more lipophilic solutes showed slower diffusivity values. The study showed that the dermis may be a much more lipophilic environment than originally believed and that distribution of smaller nonionized solutes into local tissues below a site of topical application may be estimated based on knowledge of their lipophilicity alone.

Effect of solute lipophilicity on penetration through canine skin

Objective To investigate the effect of lipophilicity on the percutaneous penetration of a homologous series of alcohols through canine skin Design Skin harvested from Greyhound thorax was placed in Franz-type diffusion cells and the in vitro passage of radio-labelled (C-14) alcohols (ethanol, butanol, hexanol and octanol (Log P 0.19 - 3.0)) through separate skin sections was measured in replicates of five. Permeability coefficient (k(P), cm/h), maximum flux (J(max), mol/cm(2)/h) and residue remaining within the skin were determined. Results The k(P) increased with increasing lipophilicity (6.2 x 10(-4) +/- 1.6 x 10(-4) cm/h for ethanol to 1.8 x 10(-2) 3.6 x 10(-3) cm/h for octanol). Alcohol residues remaining within each skin sample followed a similar pattern. An exponential decrease in Jmax with increasing lipophilicity was observed. Conclusion Changes in canine skin permeability occur with increasing alcohol lipophilicity. This finding has practical consequences for the design of topical formulations and optimisation of drug delivery through animal skin.