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Ocular syphilis is a serious complication of Treponema pallidum infection that can occur at any stage of syphilis and affect any eye structure. It remains unknown if certain T. pallidum strains are associated with ocular infections; therefore, we performed genotyping and whole genome sequencing (WGS) to characterize strains from patients with ocular syphilis. Seventy-five ocular or non-ocular specimens from 55 ocular syphilis patients in 14 states within the United States were collected between February 2016 and November 2020. Sufficient T. pallidum DNA was available from nine patients for genotyping and three for WGS. Genotyping was done using the augmented Centers for Disease Control and Prevention typing scheme, and WGS was performed on Illumina platforms. Multilocus sequence typing allelic profiles were predicted from whole genome sequence data. T. pallidum DNA was detected in various specimens from 17 (30.9%) of the 55 patients, and typing was done on samples from 9 patients. Four complete strain types (14d10/g, 14b9/g, 14d9/g, and 14e9/f) and five partial types were identified. WGS was successful on samples from three patients and all three strains belonged to the SS14 clade of T. pallidum . Our data reveal that multiple strain types are associated with ocular manifestations of syphilis. While genotyping and WGS were challenging due to low amounts of T. pallidum DNA in specimens, we successfully performed WGS on cerebrospinal fluid, vitreous fluid, and whole blood.IMPORTANCESyphilis is caused by the spirochete Treponema pallidum . Total syphilis rates have increased significantly over the past two decades in the United States, and the disease remains a public health concern. In addition, ocular syphilis cases has also been on the rise, coinciding with the overall increase in syphilis rates. We conducted a molecular investigation utilizing traditional genotyping and whole genome sequencing over a 5-year period to ascertain if specific T. pallidum strains are associated with ocular syphilis. Genotyping and phylogenetic analysis show that multiple T. pallidum strain types are associated with ocular syphilis in the United States., Competing Interests: The authors declare no conflict of interest.

Epithelial cells secrete apical extracellular matrices to form protruding structures such as denticles, ridges, scales, or teeth. The mechanisms that shape these structures remain poorly understood. Here, we show how the actin cytoskeleton and a provisional matrix work together to sculpt acellular longitudinal alae ridges in the cuticle of adult C. elegans. Transient assembly of longitudinal actomyosin filaments in the underlying lateral epidermis accompanies deposition of the provisional matrix at the earliest stages of alae formation. Actin is required to pattern the provisional matrix into longitudinal bands that are initially offset from the pattern of longitudinal actin filaments. These bands appear ultrastructurally as alternating regions of adhesion and separation within laminated provisional matrix layers. The provisional matrix is required to establish these demarcated zones of adhesion and separation, which ultimately give rise to alae ridges and their intervening valleys, respectively. Provisional matrix proteins shape the alae ridges and valleys but are not present within the final structure. We propose a morphogenetic mechanism wherein cortical actin patterns are relayed to the laminated provisional matrix to set up distinct zones of matrix layer separation and accretion that shape a permanent and acellular matrix structure., Competing Interests: The authors have declared that no competing interests exist.

Downstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5'-C-phosphate-G-3' (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now. IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum . Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

Background: The Aptima Combo 2 (AC2) assay manufactured by Hologic, Inc., detects Neisseria gonorrhoeae and/or Chlamydia trachomatis (CT) in urogenital and extragenital specimens by targeting either a 16S rRNA (N. gonorrhoeae) or 23S rRNA (CT) region. In 2019, a mutation (C1515T) in the 23S rRNA region was reported to cause false-negative/equivocal results in specimens collected in Finland. Specimens containing this variant (Fl-nvCT) were also discovered internationally. Working with specimens submitted to a large commercial laboratory, we sought to determine if this variant was also present in the United States., Methods: A subset (n = 401) of specimens tested with the AC2 assay collected during a 5-week period in late 2019/early 2020 were evaluated using an updated AC2 assay., Results: Although the FI-nvCT variant was not detected within this specimen panel, 2 CT variants containing 23S rRNA mutations (A1518G, G1526A) were identified. The updated AC2 assay targeting an additional region of the 23S rRNA detected both of these variants. A retrospective study of >18 million AC2 results tested between 2018 and 2019 did not display a decrease in CT positivity., Conclusions: Although we did not detect the Fl-nvCT variant among US specimens, we show evidence that the low occurrence of similar diagnostic-escape mutants can be detected with an updated AC2 assay using multiple 23S rRNA targets., Competing Interests: Conflict of Interest and Sources of Funding: None of the authors have conflicts of interest to disclose. This publication was funded by the Centers for Disease Control and Prevention., (Copyright © 2022 American Sexually Transmitted Diseases Association. All rights reserved.)

Abstract: The frequency of lymphogranuloma venereum or invasive Chlamydia trachomatis infection with serovar L1, L2, or L3 is unknown in the United States. While no diagnostic test is commercially available, we used a laboratory-developed test and detected lymphogranuloma venereum-associated serovar L2 in 14% of 132 remnant C. trachomatis-positive rectal swabs., Competing Interests: Conflict of Interest and Sources of Funding: None of the authors have conflicts of interest. This publication was funded by the CDC and in part supported by Cooperative Agreement No. 5NU60OE000103 between the CDC and the Association of Public Health Laboratories., (Copyright © 2021 Written work prepared by employees of the Federal Government as part of their official duties is, under the U.S. Copyright Act, a "work of the United States Government" for which copyright protection under Title 17 of the United States Code is not available. As such, copyright does not extend to the contributions of employees of the Federal Government.)

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method., Competing Interests: Declaration of competing interest The authors report no conflicts of interest relevant to this article., (Published by Elsevier Inc.)

Purpose: The LI-RADS Treatment Response (LR-TR) algorithm was introduced in 2017 to assist radiologists in assessing hepatocellular carcinoma (HCC) response following locoregional therapy. The objective of this study was to evaluate the associations between pre-treatment LI-RADS diagnostic categories, post-treatment LR-TR categories, and mRECIST response categories with overall survival (OS) of patients with HCC., Methods: This retrospective study included untreated patients with one or two lesions who underwent transarterial embolization with or without concomitant ablation from December 2003 to December 2017. Two radiologists (R1 and R2) reviewed pre- and post-treatment CT imaging. Associations between pre- and post-treatment variables, including post-treatment LR-TR categories (Viable, Equivocal, Nonviable), with OS were assessed using the Kaplan-Meier method and Cox proportional hazards regression., Results: Eighty-five patients were included (median age = 71 years, range 50-87; 17 women). The median OS from first embolization was 43.92 months. Pre- and post-treatment tumor size, pre-treatment LR-TIV (compared with LR-5), and post-treatment LR-TR Viable (compared with LR-TR Nonviable) were associated with OS (p < 0.05 for all). Median OS was shorter for LR-TR Viable patients (R1, 25.64 months, 95% CI 18.58-35.70; R2, 26.43 months 95% CI 20.68-43.92) than for LR-TR Nonviable patients (64.21 months R1 and R2, 95% CI 42.71-92.45 and 36.30-94.09, respectively). mRECIST categories showed similar associations with OS. Inter-reader agreement was moderate for LI-RADS categories (κ = 0.57, 95% CI 0.35-0.78) and substantial for LR-TR categories (κ = 0.68, 95% CI 0.55-0.81)., Conclusions: LR-TR categories show a strong association with OS in HCC patients treated with transarterial embolization., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

We reviewed the relevant syphilis diagnostic literature to address the following question: what are the performance characteristics, stratified by the stage of syphilis, for nontreponemal serologic tests? The database search included key terms related to syphilis and nontreponemal tests from 1960-2017, and for data related to the venereal disease research laboratory test from 1940-1960. Based on this review, we report the sensitivity and specificity for each stage of syphilis (primary, secondary, early latent, late latent, or unknown duration; tertiary as well as neurosyphilis, ocular syphilis, and otic syphilis). We also report on reactive nontreponemal tests in conditions other than syphilis, false negatives, and automated nontreponemal tests. Overall, many studies were limited by their sample size, lack of clearly documented clinical staging, and lack of well-defined gold standards. There is a need to better define the performance characteristics of nontreponemal tests, particularly in the late stages of syphilis, with clinically well-characterized samples. Published data are needed on automated nontreponemal tests. Evidence-based guidelines are needed for optimal prozone titrations. Finally, improved criteria and diagnostics for neurosyphilis (as well as ocular and otic syphilis) are needed., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Direct detection methods for Treponema pallidum include dark-field microscopy (DFM), direct fluorescence antibody (DFA) testing, immunohistochemistry (IHC), and nucleic acid amplification tests (NAATs). Here, we reviewed the relevant syphilis diagnostic literature to address 2 main questions with respect to T. pallidum direct detection techniques: "What are the performance characteristics for each direct detection test for T. pallidum and what are the optimal specimen types for each test?" and "What options are available for T. pallidum molecular epidemiology?" To answer these questions, we searched 5 electronic databases (OVID Medline, OVID Embase, CINAHL, Cochrane Library, and Scopus) from 1964 to 2017 using relevant search terms and identified 1928 articles, of which 37 met our inclusion criteria. DFM and DFA sensitivities ranged from 73% to 100% in cases of primary syphilis; and while sensitivity using silver stain histopathology for T. pallidum was generally low (0%-41%), higher performance characteristics were observed for T. pallidum-specific IHC (49-92%). Different genes have been targeted by T. pallidum-specific NAATs, with the majority of studies indicating that sensitivity is primarily dependent on the type of collected biological sample, with highest sensitivity observed in primary lesion exudate (75-95%). Given the rising incidence of syphilis, the development of direct, Food and Drug Administration-cleared T. pallidum NAATs should be considered an immediate priority., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

Clinical isolates of Treponema pallidum subspecies pallidum (T. pallidum) would facilitate study of prevalent strains. We describe the first successful rabbit propagation of T. pallidum from cryopreserved ulcer specimens. Fresh ulcer exudates were collected and cryopreserved with consent from syphilis-diagnosed patients (N = 8). Each of eight age-matched adult male rabbits were later inoculated with a thawed specimen, with two rabbits receiving 1.3 ml intratesticularly (IT), and six receiving 0.6 ml intravenously (IV) and IT. Monitoring of serology, blood PCR and orchitis showed that T. pallidum grew in 2/8 rabbits that were inoculated IV and IT with either a penile primary lesion specimen (CDC-SF003) or a perianal secondary lesion specimen (CDC-SF007). Rabbit CDC-SF003 was seroreactive by T. pallidum Particle Agglutination (TP-PA) and Rapid Plasma Reagin (RPR) testing, PCR+, and showed orchitis by week 6. Euthanasia was performed in week 7, with treponemal growth in the testes confirmed and quantified by qPCR and darkfield microscopy (DF). Serial passage of the extract in a second age-matched rabbit also yielded treponemes. Similarly, rabbit CDC-SF007 showed negligible orchitis, but was seroreactive and PCR+ by week 4 and euthanized in week 6 to yield T. pallidum, which was further propagated by second passage. Using the 4-component molecular typing system for syphilis, 3 propagated strains (CDC-SF003, CDC-SF007, CDC-SF008) were typed as 14d9f, 14d9g, and 14d10c, respectively. All 3 isolates including strain CDC-SF011, which was not successfully propagated, had the A2058G mutation associated with azithromycin resistance. Our results show that immediate cryopreservation of syphilitic ulcer exudate can maintain T. pallidum viability for rabbit propagation., Competing Interests: We have the following interests: Patricia Atkins [PA] is employed by Charles River Laboratories. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Purpose: Adrenocortical carcinomas (ACC) are rare and aggressive malignancies with limited treatment options. This study was undertaken to evaluate the immunogenicity of ACC., Patients and Methods: Patients with advanced ACC were enrolled in a phase II study to evaluate the clinical activity of pembrolizumab 200 mg every 3 weeks, without restriction on prior therapy. The primary end point was objective response rate. Efficacy was correlated with tumor programmed death-ligand 1 expression, microsatellite-high and/or mismatch repair deficient (MSI-H/MMR-D) status, and somatic and germline genomic correlates., Results: We enrolled 39 patients with advanced ACC and herein report after a median follow-up of 17.8 months (range, 5.4 months to 34.7 months). The objective response rate to pembrolizumab was 23% (nine patients; 95% CI, 11% to 39%), and the disease control rate was 52% (16 patients; 95% CI, 33% to 69%). The median duration of response was not reached (lower 95% CI, 4.1 months). Two of six patients with MSI-H/MMR-D tumors responded. The other seven patients with objective responses had microsatellite stable tumors. The median progression-free survival was 2.1 months (95% CI, 2.0 months to 10.7 months), and the median overall survival was 24.9 months (95% CI, 4.2 months to not reached). Thirteen percent of patients (n = 5) had treatment-related grade 3 or 4 adverse events. Tumor programmed death-ligand 1 expression and MSI-H/MMR-D status were not associated with objective response., Conclusion: MSI-H/MMR-D tumors, for which pembrolizumab is a standard therapy, are more common in ACC than has been recognized. In advanced ACC that is microsatellite stable, pembrolizumab provided clinically meaningful and durable antitumor activity with a manageable safety profile.

A guanine mononucleotide repeat in the rpsA (tp0279) gene was evaluated for improved strain discrimination using 72 Treponema pallidum-positive specimens. The tandem repeat combined with the enhanced Centers for Disease Control and Prevention typing system resulted in increased discrimination and should be useful for molecular epidemiologic studies on syphilis especially in outbreaks and among men who have sex with men.

Yaws is a neglected tropical disease caused by the bacterium Treponema pallidum subspecies pertenue. The disease primarily affects children under 15 years of age living in low socioeconomic conditions in tropical areas. As a result of a renewed focus on the disease owing to a recent eradication effort initiated by the World Health Organization, we have evaluated a typing method, adapted from and based on the enhanced Centers for Disease Control and Prevention typing method for T. pallidum subsp. pallidum, for possible use in epidemiological studies. Thirty DNA samples from yaws cases in Vanuatu and Ghana, 11 DNA samples extracted from laboratory strains, and 3 published genomic sequences were fully typed by PCR/RFLP analysis of the tpr E, G, and J genes and by determining the number of 60-bp repeats within the arp gene. Subtyping was performed by sequencing a homonucleotide "G" tandem repeat immediately upstream of the rpsA gene and an 84-bp region of tp0548. A total of 22 complete strain types were identified; two strain types in clinical samples from Vanuatu (5q11/ak and 5q12/ak), nine strain types in clinical samples from Ghana (3q12/ah, 4r12/ah, 4q10/j, 4q11/ah, 4q12/ah, 4q12/v, 4q13/ah, 6q10/aj, and 9q10/ai), and twelve strain types in laboratory strains and published genomes (2q11/ae, 3r12/ad, 4q11/ad, 4q12/ad, 4q12/ag, 4q12/v, 5r12/ad, 6r12/x, 6q11/af, 10q9/r, 10q12/r, and 12r12/w). The tpr RFLP patterns and arp repeat sizes were subsequently verified by sequencing analysis of the respective PCR amplicons. This study demonstrates that the typing method for subsp. pallidum can be applied to subsp. pertenue strains and should prove useful for molecular epidemiological studies on yaws., Competing Interests: The authors have declared that no competing interests exist.

Among men who have sex with men (MSM), those with a diagnosis of syphilis or other rectal sexually transmitted infections (STIs) are at a higher risk for human immunodeficiency virus acquisition, which is concerning given the large increase in recently reported syphilis cases in the United States. We have developed the first nonhuman primate model for rectally transmitted syphilis by exposing simian/human immunodeficiency virus-infected and naive rhesus macaques to Treponema pallidum in the rectum. All animals showed mucosal lesions, systemic dissemination, and seroconversion (treponemal antibodies). This model would be valuable for studying the manifestations of and interventions for T. pallidum infection, with and without human immunodeficiency virus coinfection.

Haemophilus ducreyi causes chancroid and has recently been shown to be a significant cause of cutaneous lesions in tropical or subtropical regions where yaws is endemic. Here, we report the draft genome assemblies for 11 cutaneous strains of Haemophilus ducreyi, isolated from children in Vanuatu and Ghana., (Copyright © 2016 Pillay et al.)

Purpose: The aim of this study was to assess the diagnostic performance of computed tomography (CT) for initial staging of non-endometrioid carcinomas of the uterine corpus., Materials and Methods: Waiving informed consent, the Institutional Review Board approved this Health Insurance Portability and Accountability Act (HIPAA)-compliant retrospective study of 193 women with uterine papillary serous carcinomas, clear cell carcinomas, and carcinosarcomas, who underwent surgical staging between May 1998 and December 2011 and had preoperative CT within 6 weeks before surgery. Two radiologists (R1, R2) independently reviewed all CT images. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and area under the curve were calculated using operative notes and surgical pathology as the reference standard., Results: The respective sensitivities and specificities achieved by R1/R2 were 0.79/0.64 and 0.87/0.75 for detecting deep myometrial invasion (MI) on CT; 0.56/0.63 and 0.93/0.79 for detecting cervical stromal invasion; 0.52/0.45 and 0.95/0.93 for detecting pelvic nodal metastases; and 0.45/0.30 and 0.98/0.98 for detecting para-aortic nodal metastases. Although CT had suboptimal sensitivity for the detection of omental disease, it had high PPV for omental seeding at surgical exploration (1.00 for R1 and 0.92 for R2). Inter-observer agreement ranged from moderate in the detection of deep MI (κ = 0.42 ± 0.06) to almost perfect in the detection of para-aortic nodal metastases (κ = 0.88 ± 0.08)., Conclusion: In patients with uterine non-endometrioid carcinomas, CT is only moderately accurate for initial staging but may provide clinically valuable information by 'ruling-in' isolated para-aortic lymph node metastases and omental dissemination.

Purpose: To evaluate interreader and inter-test agreement in applying size- and necrosis-based response assessment criteria after transarterial embolization (TAE) for hepatocellular carcinoma (HCC), applying two different methods of European Association for the Study of the Liver (EASL) criteria., Methods: Seventy-four patients (median age, 67 years) from a prospectively accrued study population were included in this retrospective study. Four radiologists independently evaluated CT data at 2-3 (1st follow-up, FU) and 10-12 (2nd FU) weeks after TAE and assessed treatment response using size-based (WHO, RECIST) and necrosis-based (mRECIST, EASL) criteria. Enhancing tissue was bidimensionally measured (EASLmeas) and also visually estimated (EASLest). Interreader and inter-test agreements were assessed using intraclass correlation coefficient (ICC) and κ statistics., Results: Interreader agreement for all response assessment methods ranged from moderate to substantial (κ = 0.578-0.700) at 1st FU and was substantial (κ = 0.716-0.780) at 2nd FU. Inter-test agreement was substantial between WHO and RECIST (κ = 0.610-0.799, 1st FU; κ = 0.655-0.782, 2nd FU) and excellent between EASLmeas and EASLest (κ = 0.899-0.918, 1st FU; κ = 0.843-0.877, 2nd FU)., Conclusion: Size- and necrosis-based criteria both show moderate to excellent interreader agreement in evaluating treatment response after TAE for HCC. Inter-test agreement regarding EASLmeas and EASLest was excellent, suggesting that either may be used., Key Points: • Applying EASL criteria, visual estimation and bidimensional measurements show comparable interreader agreement. • EASL meas and EASL est show substantial interreader agreement for treatment response in HCC. • Agreement was excellent for EASL meas and EASL est after TAE of HCC. • Visual estimation of enhancement is adequate to assess treatment response of HCC.

Purpose: To develop a preoperative CT-based nomogram for predicting overall survival (OS) in patients with non-endometrioid carcinomas of the uterine corpus., Methods: Waiving informed consent, the institutional review board approved this HIPAA-compliant, retrospective study of 193 women with histopathologically proven uterine papillary serous carcinomas (UPSC), uterine clear cell carcinomas (UCCC), and uterine carcinosarcomas (UCS) who underwent primary surgical resection between May 1998 and December 2011, and had a preoperative CT ≤ 6 weeks before surgery. All CT scans were reviewed for local or/and regional tumor extent, presence of pelvic or/and para-aortic adenopathy, and presence of distant metastases. Univariate survival analysis was performed using log-rank test and Cox regression. Variables shown significant by the univariate analysis were evaluated with the multivariable Cox regression analysis and the results were used to create a nomogram for predicting OS. The predictive accuracy of the nomogram was assessed with the concordance probability index (c-index) and a 3-year calibration plot., Results: Mean patient age was 67.2 years (range 49.0-85.9); histologies included UPSC (n = 116), UCCC (n = 27), and UCS (n = 50). Median follow-up was 38.1 months (0.9-168.5 months). At multivariate analysis, patient age, ascites, and omental implants on CT were significant adverse predictors of OS and were used to build the nomogram. Concordance index for the nomogram was 0.640 ± 0.028., Conclusion: We developed a nomogram with a good concordance probability at predicting OS based on readily available pretreatment clinical and imaging characteristics. This preoperative nomogram has the potential to improve initial treatment planning and patient counseling.

Background: Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin?, Methodology/principal Findings: To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin., Conclusions/significance: These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

Objective: The purpose of this article is to review imaging workup of perihilar cholangiocarcinoma, including MDCT and MRI protocols, imaging findings, differential diagnosis, and staging. A reporting template is included., Conclusion: Imaging plays a central role in the detection, differential diagnosis, and staging of perihilar cholangiocarcinoma.

Intraductal papillary mucinous neoplasms (IPMN) are being diagnosed with increasing frequency, necessitating an algorithm to help stratify patients into low- and high-risk groups, for follow-up versus more invasive evaluation. New evidence concerning their natural history and overall risk of malignancy has emerged since the 2006 International Association of Pancreatology consensus guidelines, prompting an update in 2012, that distinguishes radiologic 'worrisome features' from 'high-risk stigmata'. The aim of this article is to illustrate, with case examples, the variable imaging patterns of IPMN and how their radiologic features, such as cyst size and mural nodules, are interpreted in the context of the new 2012 guidelines. The 2012 and 2006 guidelines will be compared and discussed with reference to additional studies that have since been published. Despite these guidelines, lingering uncertainty remains about the natural history of IPMN, a source of unease to both radiologists and referring clinicians alike, mandating further refinement of clinical and radiologic parameters predictive of malignancy. Emerging data regarding the risk of extrapancreatic malignancy, as well as synchronous or metachronous pancreatic ductal adenocarcinoma remote in location from a branch duct IPMN are also reviewed. With the expanding research and evolving understanding of this clinicopathologic entity across the globe, radiologists will continue to play an important role in the management of patients with IPMN.

Objective: The purpose of this retrospective study was to measure interobserver agreement in the assessment of malignant imaging features of intraductal papillary mucinous neoplasms (IPMNs) on MDCT., Materials and Methods: Pancreatic protocol CT studies were reviewed for 84 patients with resected IPMNs. Maximal diameter of the dominant cyst, presence of a mural nodule, presence of a solid component, and diameters of the main pancreatic duct (MPD) and common bile duct (CBD) were measured by four radiologists independently. In each patient, the IPMN was classified into one of three types: main duct, branch duct, or mixed IPMN. Interobserver agreement of lesion features was examined using the intraclass correlation coefficient (ICC) for continuous features and Fleiss kappa for categorical features., Results: The final dataset included 55 branch duct IPMNs, nine main duct IPMNs, and 20 mixed IPMNs. Moderate agreement (ĸ = 0.458; 95% CI, 0.345-0.564) was observed in assigning branch duct, main duct, or mixed IPMN subtypes. Measurement agreement was substantial to excellent for dominant cyst (ICC = 0.852; 95% CI, 0.777-0.907), MPD (0.753, 0.655-0.837), and CBD (0.608, 0.463-0.724) but only fair to moderate for the detection of the presence of mural nodule (ĸ = 0.284, 0.125-0.432) or solid component (ĸ = 0.405, 0211-0.577)., Conclusion: Substantial to excellent interobserver agreement in the measurement of cyst diameter, MPD, and CBD support their use for characterizing malignant features of IPMN on MDCT. However, the subjective interpretation of the presence of solid components and mural nodules by individual radiologists was more variable.

Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

Human participants searched in a dynamic three-dimensional computer-generated virtual-environment open-field search task for four hidden goal locations arranged in a diamond configuration located in a 5 × 5 matrix of raised bins. Participants were randomly assigned to one of two groups: visual pattern or visual random. All participants experienced 30 trials in which four goal locations maintained the same spatial relations to each other (i.e., a diamond pattern), but this diamond pattern moved to random locations within the 5 × 5 matrix from trial to trial. For participants in the visual pattern group, four locations were marked in a distinct color and arranged in a diamond pattern that moved to a random location independent of the hidden spatial pattern from trial to trial throughout the experimental session. For participants in the visual random group, four random locations were marked with a distinct color and moved to random locations independent from the hidden spatial pattern from trial to trial throughout the experimental session. As a result, the visual cues for the visual pattern group were consistent but not coincident with the hidden spatial pattern, whereas the visual cues for the visual random group were neither consistent nor coincident with the hidden spatial pattern. Results indicated that participants in both groups learned the spatial configuration of goal locations and that the presence of consistent but noncoincident visual cues facilitated the learning of spatial relations among locations.

Background: This study evaluated the addition of sorafenib to gemcitabine and cisplatin in biliary adenocarcinoma first-line therapy., Methods: Patients with advanced biliary adenocarcinomas received gemcitabine 1000 mg m(-2) and cisplatin 25 mg m(-2) on a 2 weeks on/1 week off cycle and sorafenib 400 mg twice daily. After the initial 16 patients were enrolled, the chemotherapy doses were amended in view of grade 3 and 4 hand-foot skin reaction and haematologic toxicity. Subsequently, 21 patients received gemcitabine 800 mg m(-2), cisplatin 20 mg m(-2) and sorafenib 400 mg. The primary end point was an improvement in 6-month progression-free survival (PFS6) from historical 57-77% (90% power, type I error of 10%). Pretreatment pERK, evaluated by immunostaining, was correlated with clinical outcome., Results: A total of 39 patients were accrued. The most common grade 3-4 toxicities noted in >10% of patients were fatigue, elevated liver function tests and haematologic toxicities including thromboemboli, hyponatraemia and hypophosphataemia. Six-month progression-free survival was 51% (95% confidence interval (CI) 34-66%). Median PFS and overall survival were 6.5 (95% CI: 3.5-8.3) and 14.4 months (95% CI: 11.6-19.2 months), respectively. No correlation was observed between pERK and outcomes., Conclusion: The addition of sorafenib to gemcitabine and cisplatin in biliary adenocarcinomas did not improve efficacy over historical data, and toxicity was increased.

Changes in the osmolality and level of angiotensin II (ANG II) are important peripheral signals modulating appropriate central sympathetic output and maintaining a normal arterial pressure during high salt intake. The median preoptic nucleus (MnPO) receives reciprocal inputs from the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), the circumventricular organs that have been shown to be necessary in multiple central effects of changes in the osmolality and circulating ANG II directed toward the maintenance of sodium and water homeostasis. We, therefore, hypothesized that the MnPO is a crucial part of the central neuronal mechanisms mediating the blood pressure control by altered osmolality and/or ANG II signaling during chronic high dietary salt intake. Male Sprague-Dawley rats were randomly assigned to either sham (operation), or electrolytic lesion of the MnPO. After a 7-day recovery, rats were instrumented with radiotelemetric transducers and aortic flow probes for the measurement of the mean arterial pressure + heart rate (HR) and cardiac output (CO), respectively. Femoral venous catheters were also implanted to collect blood for the measurements of plasma osmolality and sodium concentration, as well as plasma renin activity. Rats were given another 10 days to recover and then were subjected to a 28-day-long study protocol that included a 7-day control period (1.0% NaCl diet), followed by 14 days of high salt (4.0% NaCl), and a 7-day recovery period (1.0% NaCl). The data showed, that despite a slight increase in the MAP observed in both MnPO- ( n = 12) and sham-lesioned ( n = 8) rats during the high-salt period, there were no significant differences between the MAP, HR, and CO in the two groups throughout the study protocol. These findings do not support the hypothesis that the MnPO is necessary to maintain normal blood pressure during high dietary salt intake. However, MnPO-lesioned rats showed less sodium balance than sham-lesioned rats during the first 4 days of high salt intake. Although, these results may be explained partly by the plasma hyperosmolarity and hypernatremia observed in MnPO-lesioned rats; they also shed light on the role of the MnPO in central neuronal control of renal sodium handling during chronic high dietary salt intake.

1. We have shown previously that the chronic hypotensive effect of the angiotensin II AT1 receptor antagonist losartan is mediated, in part, by the subfornical organ (SFO). However, the neural pathway(s) mediating this central effect of losartan downstream from the SFO has not been completely elucidated. 2. The present study was designed to test the hypothesis that the median preoptic nucleus (MnPO) is a crucial part of the neural pathway necessary for the chronic hypotensive effect of losartan. To test this hypothesis, male Sprague-Dawley rats were subjected to either Sham or electrolytic lesion of the MnPO (MnPOx). Rats were instrumented with radiotelemetric transducers and aortic flow probes for the continuous measurement of mean arterial pressure (MAP) and heart rate and cardiac output (CO), respectively. Total peripheral resistance (TPR) was calculated as MAP/CO. After 3 days of baseline measurements, rats were infused intraperitoneally with losartan (10 mg/kg per day) via an osmotic minipump at a rate of 5 microL/min. 3. The data revealed that, by Day 9 of losartan treatment, MAP had decreased 34 +/- 2 mmHg in MnPOx rats (n = 9), whereas the MAP of Sham-lesioned rats (n = 8) had only decreased 24 +/- 3 mmHg. These findings were accompanied by a greater decrease in TPR in MnPOx compared with Sham rats (-0.464 vs-0.237 mmHg/mL per min, respectively), whereas CO remained unchanged throughout the study protocol. 4. These results do not support the hypothesis that an intact MnPO is necessary to mediate the full chronic hypotensive effect of losartan in normal rats. Instead, they appear to suggest that the MnPO may play an important role in buffering the profound hypotension induced by losartan.

Purpose: To prospectively compare thin-section low-dose multi-detector row computed tomographic (CT) colonography with conventional colonoscopy for the detection of colorectal neoplasms., Materials and Methods: One hundred five patients underwent CT colonography immediately before colonoscopy. Supine and prone CT colonographic acquisitions to image the region during a 30-second breath hold were performed. CT colonographic images were prospectively interpreted for the presence, location, size, and morphologic features of polyps. The time of image interpretation was noted. Sensitivity, specificity, and positive and negative predictive values of CT colonography were calculated, with 95% CIs, by using colonoscopic findings as the reference standard. The weighted CT dose index was calculated on the basis of measurements in a standard body phantom. Effective dose was calculated by using commercially available software., Results: Median CT data interpretation time was 12 minutes. One hundred thirty-two polyps in 59 patients were identified at colonoscopy; no polyps were detected in 46 patients. Sensitivities for detection of polyps smaller than 5 mm, 6-9 mm, and larger than 10 mm in diameter were 12% (11 of 91 polyps), 70% (19 of 27 polyps), and 93% (13 of 14 polyps), respectively. Estimated overall specificity was 97.7% (515 of 527 imaging results). The total weighted CT dose index for combined supine and prone CT colonography was 11.4 mGy. The effective doses for combined CT colonography were 5.0 mSv and 7.8 mSv for men and women, respectively., Conclusion: Low-dose multi-detector row CT colonography has excellent sensitivity and specificity for detection of colorectal neoplasms 10 mm and larger., (Copyright RSNA, 2002)

Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, approximately 600 nM) but it was approximately 50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p ( approximately 20 nM) or BPI ( approximately 50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14(+) MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites.

How invading microorganisms are detected by the host has not been well defined. We have compared the abilities of Escherichia coli and lipopolysaccharides (LPS) purified from these bacteria to prime isolated neutrophils for phorbol myristate acetate-stimulated arachidonate release, to trigger respiratory burst in 1% blood, and to increase steady-state levels of tumor necrosis factor alpha mRNA in whole blood. In all three assays, bacteria were > or = 10-fold more potent than equivalent amounts of LPS and could trigger maximal cellular responses at ratios as low as one bacterium per 20 to 200 leukocytes. Both E. coli and LPS-triggered responses were enhanced by LPS-binding protein and inhibited by an anti-CD14 monoclonal antibody and the bactericidal/permeability-increasing protein (BPI). However, whereas O polysaccharide did not affect the potency of isolated LPS, intact E. coli carrying long-chain LPS (O111:B4) was less potent than rough E. coli (J5). Furthermore, material collected by filtration or centrifugation of bacteria incubated under conditions used to trigger arachidonate release or chemiluminescence was 5- or 30-fold less active, respectively, than whole bacterial suspensions. Extracellular BPI (not bound to bacteria) inhibited bacterial signalling, but BPI bound to bacteria was much more potent. Taken together, these findings indicate that E. coli cells can strongly signal their presence to human leukocytes not only by shedding LPS into surrounding fluids but also by exposing endotoxin at or near their surface during direct interaction with host cells.

A program of antimicrobial prophylaxis for neurosurgical patients was implemented at three community hospitals after review of the previously recorded cases of postoperative sepsis and the antibiotic sensitivity of the prevailing pathogen, Staphylococcus. No primary wound infections occurred in a series of 1000 consecutive operations. The rational basis for prophylactic antibiotics is discussed.

The turnover of free cholesterol in atheromatous plaque lipid phases was studied in a patient undergoing peripheral vascular surgery. [14C]Cholesterol was injected intravenously 139 days prior to surgery, and [3H]cholesterol was injected 12 days pre-op. The plasma cholesterol specific radioactivity decay curves were determined from the times of isotope injection until surgery. At surgery, atheroma, skin, muscle, and tendon were obtained. Lipid phases of plaque homogenate were isolated by density gradient centrifugation. The top layer of the gradient, layer 1, contained the cholesteryl ester oil droplet phase, layer 2 was enriched in phospholipid bilayer phase, layer 3 contained cholesterol monohydrate crystals and the pellet, layer 4 had more dense plaque components such as collagen and elastin. The tissue:plasma specific radioactivity ratios on days 12 and 139 respectively were muscle, 0.86, 2.47; skin, 0.74, 1.20; tendon, 0.18, 1.45; total plaque, 0.22, 1.39; plaque layer 1, 0.31, 1.50; layer 2, 0.22, 1.53; layer 3, 0.08, 0.61; and layer 4, 0.20, 0.88. Thus, plaque atheroma, which contains physically distinct forms of cholesterol, had correspondingly different rates of cholesterol turnover. Cholesterol solubilized in liquid oil droplets (layer 1) and liquid crystalline phospholipid bilayers (layer 2) had specific radioactivity values similar to those of tendon cholesterol, and represented tissue cholesterol that was undergoing slow equilibration with the plasma cholesterol pool. Pellet cholesterol (layer 4), which is probably connective tissue-associated, had lower specific radioactivity values, well below those of plasma cholesterol even after 5 months. Crystalline cholesterol (layer 3) had the lowest specific radioactivity values of all tissues and plaque fractions. Therefore, cholesterol in the crystalline state is relatively inert. Since crystalline cholesterol can account for over 40% of plaque free cholesterol, resistance to mobilization of this lipid may be an important obstacle to plaque regression.

In 12 cases of closed head injury without fracture or hematoma, but with clinical signs of increased intracranial pressure (ICP) and brain stem compression and with computed tomographic (CT) scan evidence of cerebral edema and contusion, subgaleal shunts were inserted for immediate decompression of the ventricular system and continuous drainage of hemorrhagic cerebrospinal fluid (CSF). Three patients with fixed dilated pupils and no reflexes or spontaneous respiration on admission did not improve and expired within 72 h. Nine patients who manifested Cushing's triad (bradycardia, bradypnea, and hypertension) shortly after admission made significant recovery and underwent catheter removal 1 wk later; 8 were able to be discharged home after extended periods of physiotherapy. No complications, postoperative hemorrhage or infection, were recorded.

A young white woman who had been taking oral contraceptives for 8 yr presented with the Budd--Chiari syndrome. Stenosis of the hepatic segment of her inferior vena cava was observed and surgical correction was attempted, but she died of intravascular coagulation during the procedure. Postmortem examination showed myxoid intimal proliferation involving the hepatic, portal and femoral veins, and the inferior vena cava at the level of the stenosis. The literature describing inferior vena caval lesions causing the Budd--Chiari syndrome is reviewed, and the possible role of contraceptive steroids in the present patient is discussed.

Numerous other pathologic conditions are encountered. The relatively few maneuvers and drugs presented here were chosen as the ones most commonly used in daily practice. Cardiac auscultation is just one aspect of the cardiovascular examination; equal attention should be paid to determination of jugular venous pressure, jugular venous wave form, carotid upstroke characteristics, and to observation and palpation of the precordium. Finally, if the examiner keeps in mind the hemodynamic changes induced by the various maneuvers and pharmacologic agents while auscultating the heart, and if he understands the pathophysiology of the commonly encountered cardiac lesions, he should be able to diagnose heart disease and to evaluate more intelligently various murmurs and heart sounds without necessitating the use of expensive invasive or noninvasive procedures.