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Rationale: The pathobiology of Staphylococcus aureus in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive S. aureus (MSSA) can inefficiently degrade antistaphylococcal β-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant. Objectives: Drawing from a prospectively collected biobank, we sought to understand the prevalence, natural history, potential for transmission, and antibiotic resistance profiles among nCFB-derived MSSA isolates. Methods: All individuals attending a regional consultancy nCFB clinic with sputum collected between 1981 and 2017 were considered, and those with one or more S. aureus -positive cultures composed the cohort. Each individual's most recent biobank isolate was subjected to whole-genome sequencing (including the blaZ gene), antibacterial susceptibility testing, and comparative β-lactam testing at standard (5 × 10 5  colony-forming unit [cfu]/ml) and high (5 × 10 7  cfu/ml) inocula to assess for the IE and pronounced IE. Results: Seventy-four (35.4%) of 209 individuals had one or more sputum samples with S. aureus (68 MSSA, 6 methicillin-resistant S. aureus ). Those with S. aureus infection were more likely to be female. Among 60 of 74 MSSA isolates subjected to whole-genome sequencing, no evidence of transmission was identified, although specific multilocus sequence typing types were prevalent, including ST-1, ST-15, ST-30, and ST-45. Antibiotic resistance was uncommon, except for macrolides (∼20%). Among the 60 MSSA samples, the prevalence of IE and pronounced IE was observed to be drug specific: meropenem (0% and 0%, respectively), cefepime (3% and 5%, respectively), ceftazidime (8% and 0%, respectively), cloxacillin (12% and 0%, respectively), cefazolin (23% and 0%, respectively), and piperacillin-tazobactam (37% and 17%, respectively). The cefazolin IE was associated with blaZ type A ( P  < 0.01) and ST-30 ( P  < 0.01), whereas the piperacillin-tazobactam IE was associated with type C blaZ ( P  < 0.001) and ST-15 ( P  < 0.05). Conclusions: S. aureus infection was common, although no evidence of transmission was apparent in our nCFB cohort. Although routine susceptibility testing did not identify significant resistance, inoculum-related resistance was found to be relevant for commonly used nCFB antibiotics, including cefazolin and piperacillin-tazobactam. Given previous associations between IEs and negative patient outcomes, further work is warranted to understand how this phenotype impacts nCFB disease progression.

Emergomyces canadensis pulmonary infection was incidentally diagnosed in an asymptomatic patient suspected to have metastatic osteosarcoma. Molecular diagnosis was imperative to fungal identification given overlapping histopathological features with histoplasmosis. This report documents a case of isolated pulmonary emergomycosis in an otherwise immunocompetent patient while discussing diagnostic and management pitfalls of this emerging and underdiagnosed infection., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Human granulocytic anaplasmosis is an obligate intra-granulocytic parasite that is transmitted by Ixodes scapularis and Ixodes pacificus in North America. We report on the second laboratory-confirmed case of Anaplasma phagocytophilum acquired within the province of Alberta, Canada. A 67-year-old woman from the Edmonton health zone developed nonspecific systemic symptoms including fatigue, night sweats, myalgia, headaches, and fever 6 days after noticing a tick on her left upper arm in May of 2017 (day 0). On day 13, she was found to have thrombocytopenia. Her symptoms progressed until day 16 when she was treated empirically with doxycycline, at which time she slowly improved over the subsequent 2 months. The tick was later identified as a partially engorged female blacklegged tick, I. scapularis , and it was positive for A. phagocytophilum DNA by PCR. Anaplasma serology performed retrospectively on blood samples collected on days 13, 31, and 52 showed a greater than 4-fold increase in A. phagocytophilum (IgG titers from less than 1:64 on day 13 to 1:2048 on days 31 and 52), consistent with an acute infection. Although populations of blacklegged ticks are not yet established in Alberta, suspicion should remain for tick-borne diseases because infected ticks are introduced into the province by migrating birds. This case report highlights the need to remind physicians and other public health professionals that rare, non-endemic tick-borne diseases can occasionally occur in low-risk jurisdictions.

Shiga toxin-producing Escherichia coli (STEC) infections are the product of the interaction between bacteria, phages, animals, humans, and the environment. In the late 1980s, Alberta had one of the highest incidences of STEC infections in North America. Herein, we revisit and contextualize the epidemiology of STEC O157 human infections in Alberta for the period 2009-2016. STEC O157 infections were concentrated in large urban centers, but also in rural areas with high cattle density. Hospitalization was often required when the Shiga toxin genotype stx 2a stx 2c was involved, however, only those aged 60 years or older and infection during spring months (April to June) independently predicted that need. Since the late 1980s, the rate of STEC O157-associated hemolytic uremic syndrome (HUS) in Alberta has remained unchanged at 5.1%, despite a marked drop in the overall incidence of the infection. While Shiga toxin genotypes stx 1a stx 2c and stx 2a stx 2c seemed associated with HUS, only those aged under 10 years and infection during spring months were independently predictive of that complication. The complexity of the current epidemiology of STEC O157 in Alberta highlights the need for a One Health approach for further progress to be made in mitigating STEC morbidity., Competing Interests: The authors declare no conflicts of interest.

Objectives: To identify the β-lactamase responsible for the positive detection of carbapenemase production in four clinical isolates of Pseudomonas aeruginosa that were negative by PCR for KPC, OXA-48, NDM, VIM, IMP, GES and NMC/IMI carbapenemase genes., Methods: WGS using short-read and long-read methods was used to characterize the isolates. Bioinformatic analysis was used to identify the potential gene encoding a carbapenemase. Cloning, antimicrobial susceptibility testing and biochemical and phenotypic characterization were used to determine metallo-enzyme activity. Single-nucleotide variant (SNV) typing was used to determine strain relatedness. Conjugation experiments were used to determine transmissibility of the novel carbapenemase-encoding gene., Results: WGS analysis revealed a novel class B β-lactamase gene, blaCAM-1 (Central Alberta Metallo-β-lactamase), located in a 73 kb integrative element, named IMEPaCAM-1, in the chromosome of four clinical isolates of P. aeruginosa. The cloned blaCAM-1 gene conferred carbapenem resistance to Escherichia coli TOP10. The four isolates, which were all closely related, were from three patients, all of whom spent time in the same hospital in 2008 and/or 2009. IMEPaCAM-1 could not be transferred by conjugation., Conclusions: A novel metallo-enzyme, CAM-1, is encoded on an integrative element, IMEPaCAM-1, located in the chromosome of clinical isolates of P. aeruginosa. No additional isolates harbouring CAM-1 have been identified in Alberta since 2007., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: The aim of the present study was to obtain information about deaths due to sepsis in São Paulo from 2004 to 2009 and their relationship with geographical distribution., Methods: Causes of death, both main and secondary, were defined according to the codes of the International Classification of Disease version 10 (ICD-10) contained in the database. Sepsis, septic shock, multiple organ failure, pneumonia, urinary tract infection, peritonitis and other intraabdominal infections, skin and soft tissue infections (including surgical wound infection) and meningitis were considered as immediate cause of death or as the condition leading to the immediate cause of death related or associated to sepsis., Results: In the analyzed period, there was a 15.3% increase in the absolute number of deaths from sepsis in São Paulo. The mean number of deaths during this period was 28,472±1566. Most deaths due to sepsis and sepsis-related diseases over the studied period occurred in a hospital or health care facility, showing that most of the patients received medical care during the event that led to death. We observed a significant concentration of deaths in the most populous regions, tending more toward the center of the city., Conclusions: Georeferencing data from death certificates or other sources can be a powerful tool to uncover regional epidemiological differences between populations. Our study revealed an even distribution of sepsis all over the inhabited areas of São Paulo., (Copyright © 2016 Elsevier Editora Ltda. All rights reserved.)

Human (Homo sapiens) micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsa-miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL = unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferase-expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Cytomegalovirus (CMV) encodes multiple microRNAs. While these have been partially characterized in vitro, their relevance to clinical CMV infection has not been evaluated. We analyzed samples from a cohort of solid organ transplant patients with CMV disease (n = 245) for viral microRNA expression. Several CMV microRNAs were readily detectable in patients with CMV disease in variable relative abundance. Expression level generally correlated with DNA viral load and the absence of viral microRNA was associated with faster viral clearance. Detection of hcmv-miR-UL22A-5p at baseline independently predicted the recurrence of CMV viremia upon discontinuation of antiviral therapy (OR 3.024, 95% CI: 1.35-6.8; p = 0.007). A combination of direct mRNA targeting by the microRNA and indirect modulation of gene expression involving isoforms of the transcriptional regulator C-MYC may be responsible for the broad effects seen in the association of gene transcripts with the RNA-induced silencing complex and in global protein expression upon hcmv-miR-UL22A-5p transfection. This novel study of in vivo viral microRNA expression profiles provides unique insight into the complexity of clinical CMV infection following transplantation. We provide evidence that viral microRNAs may have complex effects on gene expression and be associated with specific virologic and clinical outcomes, and thus could be further evaluated as biomarkers., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Influenza vaccine immunogenicity is suboptimal in immunocompromised patients. However, there are limited data on the interplay of T- and B- cell responses to vaccination with simultaneous immunosuppression., Methods: We collected peripheral blood mononuclear cells from transplant recipients before and 1 month after seasonal influenza vaccination. Before and after vaccination, H1N1-specific T- and B-cell activation were quantified with flow cytometry. We also developed a mathematical model using T- and B-cell markers and mycophenolate mofetil (MMF) dosage., Results: In the 47 patients analyzed, seroconversion to H1N1 antigen was demonstrated in 34%. H1N1-specific interleukin 4 (IL-4)-producing CD4(+) T-cell frequencies increased significantly after vaccination in 53% of patients. Prevaccine expression of H1N1-induced HLA-DR and CD86 on B cells was high in patients who seroconverted. Seroconversion against H1N1 was strongly associated with HLA-DR expression on B cells, which was dependent on the increase between prevaccine and postvaccine H1N1-specific IL-4(+)CD4(+) T cells (R(2) = 0.35). High doses of MMF (≥ 2 g/d) led to lower seroconversion rates, smaller increase in H1N1-specific IL-4(+)CD4(+) T cells, and reduced HLA-DR expression on B cells. The mathematical model incorporating a MMF-inhibited positive feedback loop between H1N1-specific IL-4(+)CD4(+) T cells and HLA-DR expression on B cells captured seroconversion with high specificity., Conclusions: Seroconversion is associated with influenza-specific T-helper 2 and B-cell activation and seems to be modulated by MMF., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Monitoring of cytomegalovirus cell-mediated immunity is a promising tool for the refinement of preventative and therapeutic strategies posttransplantation. Typically, the interferon-γ response to T cell stimulation is measured. We evaluated a broad range of cytokine and chemokines to better characterize the ex vivo host-response to CMV peptide stimulation. In a cohort of CMV viremic organ transplant recipients, chemokine expression-specifically CCL8 (AUC 0.849 95% CI 0.721-0.978; p = 0.003) and CXCL10 (AUC 0.841, 95% CI 0.707-0.974; p = 0.004)-was associated with control of viral replication. In a second cohort of transplant recipients at high-risk for CMV, the presence of a polymorphism in the CCL8 promoter conferred an increased risk of viral replication after discontinuation of antiviral prophylaxis (logrank hazard ratio 3.6; 95% CI 2.077-51.88). Using cell-sorting experiments, we determined that the primary cell type producing CCL8 in response to CMV peptide stimulation was the monocyte fraction. Finally, in vitro experiments using standard immunosuppressive agents demonstrated a dose-dependent reduction in CCL8 production. Chemokines appear to be important elements of the cell-mediated response to CMV infection posttransplant, as here suggested for CCL8, and translation of this knowledge may allow for the tailoring and improvement of preventative strategies., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Objectives: We conducted a retrospective study on the impact of the empiric use of linezolid on mortality in vancomycin-resistant Enterococcus spp (VRE)-colonized hematology and hematopoietic stem cell transplantation (HSCT) patients., Methods: VRE-colonized inpatients for whom complete data were available (n=100) were divided into two groups: those who received empiric linezolid in the course of fever refractory to broad-spectrum antibiotics, replacing the glycopeptide utilized for the previous 48 h, and those who did not (control group). All patients were followed until hospital discharge or death. The impact of linezolid and risk factors for all-cause mortality were evaluated; variables with p<0.10 were analyzed in a multivariate model. A Kaplan-Meier survival analysis was done to compare survival among febrile patients colonized by VRE who received empiric linezolid with patients who did not receive linezolid., Results: Patients empirically prescribed linezolid were generally younger (median age 33 vs. 44 years; p=0.008) and more likely to be recipients of an allogeneic HSCT (24 (68.6%) vs. 24 (36.9%); p=0.009) than patients who did not receive the drug. Fourteen (21.5%) VRE bloodstream infections were diagnosed, all in patients who did not receive empiric linezolid (p=0.002). In-hospital mortality was comparable in empiric linezolid and non-linezolid users (19 (54.3%) vs. 27 (41.5%), respectively; p=0.293). The Kaplan-Meier survival analysis showed no significant difference in survival comparing the group that received linezolid to the group that did not (p=0.72). Graft-versus-host disease (GVHD; odds ratio (OR) 5.90, 95% confidence interval (CI) 1.46-23.79; p=0.012) and persistence of neutropenia (OR 6.93, 95% CI 1.72-27.94; p=0.0065) were independent predictors of all-cause in-hospital death in HSCT patients, and persistence of neutropenia in non-HSCT patients (OR 8.12, 95% CI 1.22-53.8; p=0.030)., Conclusions: The empiric use of linezolid in VRE-colonized hematology patients had no impact on mortality, which appeared rather to be associated with the persistence of neutropenia in general and GVHD in the HSCT group., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.

Background: Feedback mechanisms between interferons α and λ (IFNs) may be affected by single nucleotide polymorphisms (SNP) in interleukin 28B (IL-28B; IFN-λ3) promoter region and may influence cytomegalovirus (CMV) replication., Methods: We associated IL-28B SNPs with the risk of CMV replication after transplantation. Next, we examined the effect of IL-28B genotypes on IL-28B, and IFN-stimulated gene (ISG) expression, and CMV replication in human foreskin fibroblast (HFF) and peripheral blood mononuclear cells (PBMCs)., Results: Transplant recipients with an IL-28B SNP (rs8099917) had significantly less CMV replication (P = .036). Both HFF-cells and PBMCs with a SNP showed lower IL-28B expression during infection with CMV, but higher "antiviral" ISG expression (eg, OAS1). Fibroblasts with a SNP had a 3-log reduction of CMV replication at day 4 (P = .004). IL-28B pretreatment induced ISG expression in noninfected fibroblasts, but a relative decrease of ISG expression could be observed in CMV-infected fibroblasts. The inhibitory effects of IL-28B could be abolished by siRNA or antagonistic peptides against the IL-28 receptor. In fibroblasts, inhibition of IL-28 signaling resulted in an increase of ISG expression and 3-log reduction of CMV-replication (P = .01)., Conclusions: We postulate that IL-28B may act as a key regulator of ISG expression during primary CMV infection. IL-28B SNPs may be associated with higher antiviral ISG expression, which results in better replication control., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background & Aims: Very low density lipoproteins (VLDLs) are triacylglycerol (TG)-rich lipoproteins produced by the human liver. VLDLs derive the majority of their TG cargo from the lipolysis of TG stored in hepatocellular lipid droplets (LDs). Important roles for LDs and the VLDL secretory pathway in the cell culture production of infectious hepatitis C virus (HCV) have been established. We hypothesized that TG lipolysis and VLDL production are impaired during HCV infection so that these cellular processes can be diverted towards HCV production., Methods: We used an HCV permissive cell culture system (JFH-1/HuH7.5 cells) to examine the relationship between TG lipolysis, VLDL assembly, and the HCV lifecycle using standard biochemical approaches., Results: Lipolysis of cellular TG and VLDL production were impaired in HCV infected cells during the early peak of viral infection. This was partially explained by an apparent deficiency of a putative TG lipase, arylacetamide deacetylase (AADAC). The re-introduction of AADAC to infected cells restored cellular TG lipolysis, indicating a role for HCV-mediated downregulation of AADAC in this process. Defective lipolysis of cellular TG stores and VLDL production were also observed in HuH7.5 cells stably expressing a short hairpin RNA targeting AADAC expression, proving AADAC deficiency contributes to these defective pathways. Finally, impaired production of HCV was observed with AADAC knockdown cells, demonstrating a role for AADAC in the HCV lifecycle., Conclusions: This insight into the biology of HCV infection and possibly pathogenesis identifies AADAC as a novel and translationally relevant therapeutic target., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Aim: Cytomegalovirus is a common viral pathogen that influences the outcome of organ transplantation. To date, there is no established method to evaluate the effects of human CMV (HCMV) treatments in vivo except for human clinical trials. In the current study, we describe the development of a mouse model that supports the in vivo propagation of HCMV., Methods: One million viable human hepatocytes, purified from human livers, were injected into the spleens of severe combined immunodeficient/albumin linked-urokinase type plasminogen activator transgenic mice. A clinical strain of HCMV was inoculated in mice with confirmed human hepatocyte engraftment or in non-chimeric controls. Infection was monitored through HCMV titers in the plasma. Mice were administrated ganciclovir (50 mg/kg per day, i.p.) beginning at 2 days post-HCMV inoculation, or human liver natural killer (NK) cells (20 × 10(6)  cells/mouse, i.v.) 1 day prior to HCMV inoculation., Results: Chimeric mice that received HCMV showed high plasma titers of HCMV DNA on days 1 and 6 that became undetectable by day 11 post-inoculation. In contrast, non-transplanted mice had only residual plasma inoculum detection at day 1 and no detectable viremia thereafter. The levels of HCMV DNA were reduced by ganciclovir treatment or by human liver NK cell adoptive transfer, while HCMV-infected chimeric mice that were not treated sustained viremia during the follow up., Conclusion: Human liver chimeric mice provide an in vivo model for the study of acute HCMV infection of hepatocytes., (© 2012 The Japan Society of Hepatology.)

Background: The molecular adsorbent recirculating system (MARS) is an albumin-dialysis modality that has been investigated predominantly in patients with acute and acute-on-chronic liver failure., Objectives: To report the clinical efficacy and safety of MARS therapy for intractable pruritus in cholestasis patients with stable chronic liver disease, characterizing the impact of MARS on cytokine levels and on the transcriptome in the blood compartment., Methods: MARS therapy was performed on three patients with cholestatic liver disease using 8 h runs for two consecutive days. The expression levels of 65 cytokines⁄chemokines and 24,000 genes were profiled by Luminex (Luminex Corporation, USA) and microarray, respectively., Results: A quality-of-life assessment demonstrated a marked improvement during therapy, which was sustained in two of three patients. No bleeding or infectious complications were observed. Bile acid levels were markedly reduced following MARS (mean [± SD] pretreatment 478.9±112.2 µmol⁄L versus post-treatment 89.7±68.8 µmol⁄L). Concordant decreases in cytokine⁄chemokine levels were noted for interleukin (IL)-1beta, IL-2, IL-6, IL-8, IL-12 (p40), RANTES, tranforming growth factor-alpha, tumour necrosis factor-alpha and thrombopoietin following MARS. On microarray profiling, biologically relevant concordant changes among all patients were evident for 20 different genes (10 upregulated and 10 downregulated). The upregulation of several potentially immune suppressive⁄regulatory genes (eg, early growth response 3 [EGR-3], ephrin-A2 [EFNA2] and serum amyloid A1 [SAA1]), concurrent with downregulation of genes involved in innate immunity (eg, toll-like receptor 4 interactor with leucine-rich repeats [TRIL]) and inflammation (eg, ephrin receptor B1 [EPHB1]), was observed., Conclusions: This investigative approach offers new insights into intractable pruritus and suggests future therapeutic targets. The clinical benefit of MARS in cholestasis patients with intractable pruritus may not exclusively result from filtration of pruritogens, but also from systemic changes in cytokine⁄chemokine levels and changes in gene expression of blood cells.

Background: Cytomegalovirus (CMV) displays genetic polymorphisms in multiple genes, which may result in important virulence differences. Glycoprotein N (gN) and immediate early 1 (IE1) are key viral genes and immune targets. We aimed to characterize the molecular epidemiology of gN and IE1 genotypes in organ transplant patients with CMV disease in the context of clinical and virologic endpoints., Methods: A total of 240 patients with CMV disease had genotyping analysis by nested polymerase chain reaction assays and sequencing using blood samples obtained at disease onset. Results were correlated with viral clearance kinetics and recurrence., Results: Complex patterns of gN and IE1 genotypes were present with no clear genetic linkages. No single genotype of IE1 or gN was associated with poorer outcome. For example, different gN or IE1 genotypes had comparable baseline viral load, clearance half-lives, time to clearance, and rates of virologic recurrence. Mixed infection was present at IE1 in 15.8% and gN in 21.9%, but analysis of a single gene was insufficient to detect all mixed infections. Infections caused by multiple strains, as opposed to single strains, were associated with higher baseline viral loads (P = 0.011), delayed viral clearance (P = 0.033), and higher rates of virologic recurrence (P = 0.008)., Conclusions: Genetic diversity in CMV is complex. Specific gN or IE subtypes do not seem to affect in vivo viral virulence patterns in single-strain infections. Mixed infections demonstrate associations with virologic outcomes that single-strain infections do not., (© 2011 John Wiley & Sons A/S.)

Background: A CD8+ T-cell response to cytomegalovirus (CMV) has been associated with control of viral replication. Assessment shortly after the onset of asymptomatic viremia could help significantly refine preemptive strategies., Methods: We conducted a prospective study of organ transplant recipients who developed asymptomatic low-level viremia not initially requiring antiviral therapy. Cell-mediated immunity (CMI) was measured shortly after viremia onset and longitudinally using the Quantiferon-CMV assay. The primary outcome was the ability to predict spontaneous clearance versus virologic and/or clinical progression., Results: We enrolled 42 transplant patients, of which 37 were evaluable. Viral load at onset was 1140 copies/mL (interquartile range 655-1542). Spontaneous viral clearance occurred in 29 of 37 (78.4%) patients and 8 of 37(21.6%) had clinical and/or virologic progression requiring antivirals. At baseline, a positive CMI test (interferon-γ≥0.2 IU/mL) was present in 26 of 37(70.3%) patients. In patients with a positive CMI, the incidence of subsequent spontaneous viral clearance was 24 of 26 (92.3%) compared with 5 of 11 (45.5%) in patients with a negative CMI at onset (P=0.004). The absolute interferon-γ production was higher in patients with spontaneous clearance versus progression at all time points tested. Analysis of different cutoffs for defining a positive test suggested that the best threshold was 0.1 or 0.2 IU/mL of interferon-γ., Conclusions: CMI assessment shortly after the onset of CMV viremia may be useful to predict progression versus spontaneous viral clearance, thereby helping guide the need for antiviral therapy and refining current preemptive strategies.

Objective: To evaluate whether the Pretransplantion Assesment of Mortality risk score is associated to transplant costs and can be used not only to predict mortality but also as a cost management tool., Methods: We evaluated consecutively patients submitted to allogeneic (n = 27) and autologous (n = 89) hematopoietic stem cell-transplantation from 2004 to 2006 at Hospital Israelita Albert Einstein (SP), Brazil. Participants mean age at hematopoietic stem cell-transplantation was 42 (range 1 to 72) years; there were 69 males and 47 females; 30 patients had multiple myeloma; 41 had non-Hodgkin and Hodgkin's lymphomas; 22 had acute leukemia; 6 had chronic leukemia; and 17 had non-malignant disease. The Pretransplantion Assesment of Mortality risk score was applied in all patients using the available web site (http://cdsweb.fhcrc.org/ pam/)., Results: Patients could be classified in three risk categories: high, intermediate and low, having significant difference in survival (p = 0.0162). The median cost in US dollars for each group was $ 281.000, $ 73.300 and $ 54.400 for high, intermediate and low risk, respectively. The cost of hematopoietic stem cell-transplantation significantly differed for each Pretransplantin Assesment of Mortality risk group (p = 0.008)., Conclusion: The validation of the Pretransplantion Assesment of Mortality risk score in our patients confirmed that this system is an important tool to be used in transplantation units, being easy to apply and fully reproducible.

Background: CMV-specific T-cells are crucial to control CMV-replication post-transplant. Regulatory T-cells (T-regs) are associated with a tolerant immune state and may contribute to CMV-replication. However, T-cell subsets such as T-regs and IL-17 producing T-cells (Th-17) are not well studied in this context. We explored T-regs and Th-17 frequencies during CMV-replication after transplantation., Methods: We prospectively evaluated 30 transplant patients with CMV-viremia. We quantified CMV-specific CD4(+) and CD8(+) T-cells, T-regs (CD4(+)CD25(+)FoxP3(+)) and Th-17 frequencies using flow-cytometry and followed patients requiring anti-viral treatment. Two subsets were compared: anti-viral treatment requirement (n = 20) vs. spontaneous clearance of viremia (n = 10)., Results: Higher initial CMV-specific CD4(+) T-cells and lower T-regs were observed in patients with spontaneous clearance (p = 0.043; p = 0.021 respectively). Using a ratio of CMV-specific CD4(+) T-cells to T-regs allowed prediction of viral clearance with 80% sensitivity and 90% specificity (p = 0.001). One month after stop of treatment, the same correlation was observed in patients protected from CMV-relapse. The ratio of CMV-specific CD4(+) T-cells to T-regs allowed prediction of relapse with 85% sensitivity and 86% specificity (p = 0.004). Th-17 responses were not correlated with virologic outcomes., Conclusions: This study provides novel insights into T-regs and Th-17 subpopulations during CMV-replication after transplantation. These preliminary data suggest that measurement of CMV-specific CD4(+) T-cells together with T-regs has value in predicting spontaneous clearance of viremia and relapse.

Background: Cytomegalovirus (CMV) disease after discontinuation of prophylaxis is a significant problem for CMV-seronegative recipients of CMV-seropositive organs (donor seropositive and recipient seronegative [D+/R-]). Virologic monitoring after prophylaxis has been proposed as a way to prevent late-onset disease., Methods: We reviewed the efficacy of this strategy. CMV D+/R- organ transplant recipients received 3 to 6 months of antiviral prophylaxis, and then viral loads were performed weekly for 8 weeks. Preemptive antiviral therapy was initiated at a predefined threshold., Results: Seventy-one CMV D+/R- patients were assessed. Symptomatic CMV disease occurred in 29 of 71 (40.8%) patients during the first-year posttransplant. A significant portion of disease occurred only after the 8-week surveillance period (n=16). Viremia occurred in 19 of 71 (26.8%) patients during the 8-week surveillance. Preemptive therapy was successfully used in only 3 of 19 (15.8%) viremic patients with no further disease development. The remaining patients cleared low-level viremia spontaneously (n=3) or had CMV disease (n=13) either at the first detection of viremia or before preemptive therapy initiation because of rapid viral load doubling (median doubling time 1.1 days)., Conclusion: CMV D+/R- patients had significant incidence of late-onset disease after prophylaxis. However, the use of a preemptive after prophylaxis strategy was of limited benefit in this group because of rapid viral doubling times and disease occurring after the surveillance period.

Background: In patients with cytomegalovirus (CMV) disease, regular monitoring of viral loads and treatment until negative are recommended. However, with more sensitive polymerase chain reaction (PCR) assays and cellular peripheral sample types, detection of low-level viremia is achievable. We compared a whole blood real-time PCR with a plasma PCR assay for monitoring therapeutic response., Methods: Patients enrolled in a trial to treat CMV disease for 21 days had regular viral load monitoring. The results of a plasma-based PCR assay were compared with a real-time PCR assay of whole blood and assessed for their ability to predict recurrence., Results: In 219 evaluable patients, viral loads in plasma versus whole blood demonstrated good correlation but significant difference in absolute value and clearance kinetics. Virus was still detectable by day 21 in 154 of 219 (70.3%) patients with the whole blood versus 105 of 219 (52.1%; P<0.001) patients with the plasma assay. The positive predictive value of persistent plasma viremia at day 21 for virologic recurrence was 41.9% vs. 36.3% for the whole blood assay. In the subset of patients with a negative plasma but positive whole blood at day 21 (n = 49), the incidence of virologic recurrence was similar to that of all patients with a negative plasma assay (23.1% vs. 23.6%)., Conclusions: When treating CMV disease, enhanced detection of residual viremia using a whole blood real-time PCR does not seem to offer significant clinical advantages nor allows for better prediction of recurrence of CMV viremia or disease. The treat-to-negative paradigm may not hold true when such assays are used.

Background: Obesity is a risk factor for patients undergoing major surgery. In liver transplantation, the morbidity and mortality in these patients may be higher owing to concomitant diseases that may prolong hospital stay. Moreover, the restrictive respiratory pattern in these patients, associated with pulmonary complications related to liver disease can impact the postoperative recovery. We sought to analyze the impact of high body mass index (BMI) on hospital and intensive care unit (ICU) stay, necessity and length of use either invasive and noninvasive ventilatory support in the early postoperative period after liver transplantation., Patients and Methods: Between January 2007 and March 2009, we performed 85 liver transplantations in adult patients. BMI was calculated on the day of the transplantation. Data from 136 recipients undergoing OLT were reviewed by age, gender, etiology of liver disease, Model for End-Stage Liver Disease score, Child-Pugh class, cold and warm ischemic times, ICU stay, duration of invasive mechanical, and use of noninvasive ventilation (NIV). We divided the patients into 3 groups: Group 1, (normal weight BMI 18.5-24.99), versus group 2 overweight--BMI 25-29.99; versus group 3, obese--BMI ≥30., Results: Groups 1, 2, and 3 had similar lengths of stay in the ICU, necessity of NIV as well as 6 month, 1- and 2-year survivals (P > .05)., Conclusion: High BMI patients showed similar results to normal or overweight patients. Obesity should not be contraindication to liver transplantation., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: A few studies have performed to evaluate the temperature variation influences over on the stroke rates in Brazil., Method: 176 medical records of inpatients were analyzed after having had a stroke between 2004 and 2006 at Hospital Israelita Albert Einstein. The temperature preceding the occurrence of the symptoms was recorded, as well as the temperature 6, 12 and 24 hours before the symptoms in 6 different weather substations, closest to their houses in São Paulo., Results: Strokes occurred more frequently after a variation of 3 C between 6 and 24 hours before the symptoms. There were most hospitalizations between 23-24 C., Conclusion: Incidence of stroke on these patients was increased after a variation of 3 masculine Celsius within 24 hours before the ictus. The temperature variations could be an important factor in the occurrence of strokes in this population.

Background: Our previous results showed that nitric oxide (NO) and bradykinin (BK) mediate the arthritis induced by Bothrops jararaca venom (BjV) in rabbits. In this study, we investigated the contribution of each receptor of BK as well as the inter-relationship between NO and eicosanoids in BjV-induced arthritis., Methods: The arthritis was induced in rabbits with 16 microg of BjV injected intra-articularly. Prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4) (radioimmunoassay) and nitrite/nitrate concentrations (NO2/NO3) (Griess reaction) were evaluated in the synovial fluid 4 h later. The animals were prior treated with NO synthase inhibitor (L-NAME; 20 mg/kg/day for 14 days), the B2 antagonist of BK (HOE-140) and the B1 antagonist of BK (des-Arg9[Leu8]-bradykinin), both at a dose of 0.3mg/kg, 30 min prior to the venom injection., Results: Data show that L-NAME and HOE-140 treatment were equally able to reduce PGE2 and NO2/NO3 levels without interfering with TxB2 and LTB4 production. On the contrary, the B1 antagonist of BK inhibited TxB2 and LTB4 production, and did not alter PGE2 and NO metabolites levels in the inflamed joint., Discussions: The results presented clarify the contribution of the kinin system, mainly through the B2 receptor, to the local inflammatory response induced by BjV, as well as its positive interaction with PGE2 and NO production.