Evaluation of a laboratory-developed multiplex real-time PCR assay for diagnosis of syphilis, herpes and chancroid genital ulcers in four public health laboratories in the USA.

OBJECTIVE: To evaluate the field performance of a multiplex PCR (M-PCR) assay for detection of herpes simplex virus (HSV)-1 and HSV-2, Treponema pallidum (T. pallidum) and Haemophilus ducreyi (H. ducreyi) in genital ulcer disease (GUD) specimens. METHODS: GUD M-PCR was performed on 186 remnant specimens, previously collected for HSV testing, by four public health laboratories (PHLs) and the Laboratory Reference and Research Branch (LRRB) at the Centers for Disease Control and Prevention. The results from the PHLs were compared with those of LRRB, which served as the reference testing method, and percentage agreement was calculated. RESULTS: HSV was detected in 31 of 52 (59.6%), 20 of 40 (50%), 43 of 44 (97.7%) and 19 of 50 (38.0%) specimens from PHL1, PHL2, PHL3 and PHL4, respectively. There were seven discrepant results for HSV, and the overall percent agreement between the PHLs and the LRRB was 94%-100%, with a kappa value of 0.922, which demonstrates high agreement. T. pallidum was identified in 7 of 51 (13.7%) specimens from PHL1 with 94.1% agreement and in 2 of 40 (5.0%) specimens from PHL2 with 100% agreement. The LRRB identified three additional T. pallidum-positive specimens from PHL1. The kappa value (0.849) for T. pallidum testing suggests good agreement. Consistent with the LRRB results, no T. pallidum was detected in specimens from PHL3 and PHL4, and H. ducreyi was not detected at any of the study sites. CONCLUSIONS: The GUD M-PCR assay performed well in four independent PHLs and 12 suspected syphilis cases were identified in this study. The M-PCR assay could provide improved diagnostic options for GUD infections in state and local PHLs.

Chemistries for Making Additive Nanolithography in OrmoComp Permissive for Cell Adhesion and Growth.

Two-photon lithography allows writing of arbitrary nanoarchitectures in photopolymers. This design flexibility opens almost limitless possibilities for biological studies, but the acrylate-based polymers frequently used do not allow for adhesion and growth of some types of cells. Indeed, we found that lithographically defined structures made from OrmoComp do not support E18 murine cortical neurons. We reacted OrmoComp structures with several diamines, thereby rendering the surfaces directly permissive for neuron attachment and growth by presenting a surface coating similar to the traditional cell biology coating achieved with poly-d-lysine (PDL) and laminin. However, in contrast to PDL-laminin coatings that cover the entire surface, the amine-terminated OrmoComp structures are orthogonally modified in deference to the surrounding glass or plastic substrate, adding yet another design element for advanced biological studies.

Implementation of plasmonic band structure to understand polariton hybridization within metamaterials.

Gap surface plasmons (GSPs) serve a diverse range of plasmonic applications, including energy harvesting, communications, molecular sensing, and optical detection. GSPs may be realized where tightly spaced plasmonic structures exhibit strong spatial overlap between the evanescent fields. We demonstrate that within similar, nested geometries that the near-fields of the GSPs within the individual nanostructures are hybridized. This creates two or more distinct resonances exhibiting near-field distributions extended over adjacent spatial regions. In contrast, dissimilar, nested structures exhibit two distinct resonances with nominally uncoupled near-fields, resulting in two or more individual antenna resonance modes. We deploy plasmonic band structure calculations to provide insight into the type and degree of hybridization within these systems, comparing the individual components. This understanding can be used in the optimized design of polaritonic metamaterial structures for desired applications.

Influenza A(H3N2) Variant Virus Outbreak at Three Fairs - Maryland, 2017.

On September 17, 2017, the Maryland Department of Agriculture (MDA) was notified by fair and 4-H officials of ill swine at agricultural fair A, held September 14-17. That day, investigation of the 107 swine at fair A revealed five swine with fever and signs of upper respiratory tract illness. All five respiratory specimens collected from these swine tested positive for influenza A virus at the MDA Animal Health Laboratory, and influenza A(H3N2) virus was confirmed in all specimens by the U.S. Department of Agriculture National Veterinary Services Laboratory (NVSL). On September 18, MDA was notified by fair and 4-H officials that swine exhibitors were also ill. MDA alerted the Maryland Department of Health (MDH). A joint investigation with MDH and the local health department was started and later broadened to Maryland agricultural fairs B (September 13-17) and C (September 15-23). In total, 76 persons underwent testing for variant influenza, and influenza A(H3N2) variant (A(H3N2)v) virus infection was identified in 40 patients with exposure to swine at these fairs (Figure), including 30 (75%) who had more than one characteristic putting them at high risk for serious influenza complications; 24 (60%) of these were children aged <5 years. Twenty-six (65%) patients reported direct contact with swine (i.e., touching swine or swine enclosure), but 14 (35%) reported only indirect contact (e.g., walking through a swine barn). Two children required hospitalization; all patients recovered. This outbreak highlights the risk, particularly among children, for contracting variant influenza virus at agricultural fairs after direct or indirect swine contact. Publicizing CDC's recommendation that persons at high risk for serious influenza complications avoid pigs and swine barns might help prevent future variant influenza outbreaks among vulnerable groups (1).

Quantifying the relationship between optical anatomy and retinal physiological sensitivity: A comparative approach.

Light intensity varies 1 million-fold between night and day, driving the evolution of eye morphology and retinal physiology. Despite extensive research across taxa showing anatomical adaptations to light niches, surprisingly few empirical studies have quantified the relationship between such traits and the physiological sensitivity to light. In this study, we employ a comparative approach in frogs to determine the physiological sensitivity of eyes in two nocturnal (Rana pipiens, Hyla cinerea) and two diurnal species (Oophaga pumilio, Mantella viridis), examining whether differences in retinal thresholds can be explained by ocular and cellular anatomy. Scotopic electroretinogram (ERG) analysis of relative b-wave amplitude reveals 10- to 100-fold greater light sensitivity in nocturnal compared to diurnal frogs. Ocular and cellular optics (aperture, focal length, and rod outer segment dimensions) were assessed via the Land equation to quantify differences in optical sensitivity. Variance in retinal thresholds was overwhelmingly explained by Land equation solutions, which describe the optical sensitivity of single rods. Thus, at the b-wave, stimulus-response thresholds may be unaffected by photoreceptor convergence (which create larger, combined collecting areas). Follow-up experiments were conducted using photopic ERGs, which reflect cone vision. Under these conditions, the relative difference in thresholds was reversed, such that diurnal species were more sensitive than nocturnal species. Thus, photopic data suggest that rod-specific adaptations, not ocular anatomy (e.g., aperture and focal distance), drive scotopic thresholds differences. To the best of our knowledge, these data provide the first quantified relationship between optical and physiological sensitivity in vertebrates active in different light regimes.

Interorganizational network changes among health organizations in the Brazos Valley, Texas.

Community health development is a process by which a community identifies factors influencing population health, assesses available resources to build the capacity to plan and take action, and implement interventions to address identified needs. At its core, community health development targets structural change and infrastructure development to facilitate more efficient and effective health service delivery systems and environmental changes to support improvements in community health. One indicator of structural change and common measure of community capacity is the relationships among the network of organizations that comprise that system. The Brazos Valley has employed a community health development approach to population health improvement in partnership with the Center for Community Health Development. Changes in interorganizational networks illustrate progress in the Brazos Valley. Contextual factors provide some insight into how the process has unfolded.

Identification of an ICP27-responsive element in the coding region of a herpes simplex virus type 1 late gene.

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the cis-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a cis-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp "silencing element" (SE) in the 5' portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.

Herpes simplex virus type 1 ICP27 regulates expression of a variant, secreted form of glycoprotein C by an intron retention mechanism.

We previously showed that herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP27 can posttranscriptionally stimulate mRNA accumulation from a transfected viral late gene encoding glycoprotein C (gC) (K. D. Perkins, J. Gregonis, S. Borge, and S. A. Rice, J. Virol. 77:9872-9884, 2003). We began this study by asking whether ICP27 homologs from other herpesviruses can also mediate this activity. Although the homologs from varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) were inactive, the homolog from bovine herpesvirus 4 (BHV-4), termed HORF1/2, was a very efficient transactivator. Surprisingly, most of the mRNA produced via HORF1/2 transactivation was 225 nucleotides shorter than expected due to the removal of a previously undescribed intron from the gC transcript. We found that the gC mRNA produced in the absence of transactivation was also mostly spliced. In contrast, gC mRNA produced by ICP27 transactivation was predominantly unspliced. Based on these results, we conclude that ICP27 has two distinct effects on the transfected gC gene: it (i) stimulates mRNA accumulation and (ii) promotes the retention of an intron. Interestingly, the spliced transcript encodes a variant of gC that lacks its transmembrane domain and is secreted from transfected cells. As the gC splicing signals are conserved among several HSV-1 strains, we investigated whether the variant gC is expressed during viral infection. We report here that both the spliced transcript and its encoded protein are readily detected in Vero cells infected with three different laboratory strains of wild-type HSV-1. Moreover, the variant gC is efficiently secreted from infected cells. We have designated this alternate form of the protein as gCsec. As the extracellular domain of gC is known to bind heparan sulfate-containing proteoglycans and to inhibit the complement cascade via an interaction with complement component C3b, we speculate that gCsec could function as a secreted virulence factor.

A new technique for the surgical creation of aneurysms in an in vivo tortuous vessel model to test neurovascular devices.

The purpose of this study was to develop an aneurysm model that mimics the tortuous anatomy of the cerebrovasculature for the evaluation of endovascular devices. This model is an adaptation of the carotid siphon model of Georganos et al. The common carotid artery trunks in 10 swine were surgically elongated using an EXXCEL Soft ePTFE vascular graft and then sutured into position to form an S-curve, with each bend having a 5- to 10-mm radius. Following a 3- to 4-week healing period, aneurysms were surgically created from jugular vein grafts along or distal to the tortuous segment and immediately embolized with coils. In a subset (n = 6) of the arteries, a stent was also placed across the aneurysm neck. Animals were allowed to survive for 30 days. Clinical relevance and utility of the model were evaluated based on comparison to human angiographic images, physician feedback, and histopathological assessment. Tortuous anatomy was successfully created in all 10 animals, and aneurysms were added at various locations within or distal to the tortuous segment in a subset of 8 animals, creating 11 aneurysms in total. At 30 days, 18/20 vessels were patent and the bend radius was maintained. Endovascular access to aneurysms and placement of embolization coils and/or stents was successful in 10 of 11 attempts. Physician feedback indicated this tortuous model was more clinically relevant in terms of endovascular device delivery and deployment compared to established, nontortuous aneurysm models.

Impedance-based retinal contact imaging as an aid for the placement of high resolution epiretinal prostheses.

An important factor in effective stimulation of the retina is close contact with the retina. The design of the electrode surface and the placement of the electrode against the retina both affect the degree of contact with the retina. We have addressed the design factor by creating a curved surface 3200-electrode array. The placement factor we have addressed by use of an impedance sensitive feedback from the array. The feedback is in the form of an image showing contact with the retina, where greater pixel intensity indicates greater impedance and thus closer contact with the retina. In this paper, we present qualitative and quantitative assessments of the relationship between impedance and the device output as well as an in vivo demonstration of contact imaging. In addition, we evaluated the three-dimensional profile of the stimulation voltage distribution to assess the importance of close retinal contact for high resolution stimulation.

ICP27-dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0.

It was previously shown that herpes simplex virus type 1 (HSV-1) is sensitive to leptomycin B (LMB), an inhibitor of nuclear export factor CRM1, and that a single methionine to threonine change at residue 50 (M50T) of viral immediate-early (IE) protein ICP27 can confer LMB resistance. In this work, we show that deletion of residues 21-63 from ICP27 can also confer LMB resistance. We further show that neither the M50T mutation nor the presence of LMB affects the nuclear shuttling activity of ICP27, suggesting that another function of ICP27 determines LMB resistance. A possible clue to this function emerged when it was discovered that LMB treatment of HSV-1-infected cells dramatically enhances the cytoplasmic accumulation of two other IE proteins, ICP0 and ICP4. This effect is completely dependent on ICP27 and is reversed in cells infected with LMB-resistant mutants. Moreover, LMB-resistant mutations in ICP27 enhance the nuclear localization of ICP0 and ICP4 even in the absence of LMB, and this effect can be discerned in transfected cells. Thus, the same amino (N)-terminal region of ICP27 that determines sensitivity to LMB also enhances ICP27's previously documented ability to promote the cytoplasmic accumulation of ICP4 and ICP0. We speculate that ICP27's effects on ICP4 and ICP0 may contribute to HSV-1 LMB sensitivity.

Transactivation of a viral target gene by herpes simplex virus ICP27 is posttranscriptional and does not require the endogenous promoter or polyadenylation site.

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that stimulates viral mRNA expression from many viral delayed-early and late genes during infection. One HSV-1 late gene which is highly dependent on ICP27 during infection is that encoding the glycoprotein C (gC). Here we report that the gC gene is specifically transactivated by ICP27 in transfected Vero cells. Using various gC plasmid constructs, we show that ICP27's stimulatory effects are independent of the gC gene's endogenous promoter and polyadenylation site. This suggests that ICP27-responsive elements lie in the transcribed body of the gC gene. We also show that transactivation of the gC gene by ICP27 is independent of other viral proteins, as ICP27 alone can transactivate the gC gene when its transcription is mediated by the human cytomegalovirus immediate-early gene promoter. However, when gC gene expression is driven by its endogenous promoter, the stimulatory effect of ICP27 requires additional transactivators. To explore the level at which ICP27 transactivates the gC gene, we established stably transfected Vero cell lines that have integrated copies of the gC gene under control of the cytomegalovirus immediate-early gene promoter. These gC genes are not constitutively expressed but can be efficiently induced by HSV-1 infection. Using nuclear run-on transcription assays, we show that transcriptional induction of the stably transfected genes is ICP27 independent. In contrast, accumulation of gC mRNA is very highly dependent on ICP27. Together, these results demonstrate that ICP27 posttranscriptionally activates mRNA expression from a biologically relevant viral target gene.