Isolated Pulmonary Emergomycosis in an Immunocompetent Patient in Alberta, Canada.

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.

Staphylococcus aureus in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum ß-Lactam Resistance.

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 ß-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 ß-lactam testing at standard (5 × 105 colony-forming unit [cfu]/ml) and high (5 × 107 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.

Case Report: Anaplasmosis in Canada: Locally Acquired Anaplasma phagocytophilum Infection in Alberta.

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.

Epidemiology of Shiga Toxin-Producing Escherichia coli O157 in the Province of Alberta, Canada, 2009-2016.

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 stx2a stx2c 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 stx1a stx2c and stx2a stx2c 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.

Identification of a novel metallo-ß-lactamase, CAM-1, in clinical Pseudomonas aeruginosa isolates from Canada.

OBJECTIVES: To identify the ß-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 ß-lactamase gene, blaCAM-1 (Central Alberta Metallo-ß-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.

Empiric use of linezolid in febrile hematology and hematopoietic stem cell transplantation patients colonized with vancomycin-resistant Enterococcus spp.

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.

Effect of Immunosuppression on T-Helper 2 and B-Cell Responses to Influenza Vaccination.

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.

IL-28B is a key regulator of B- and T-cell vaccine responses against influenza.

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.

Immunomodulatory Function of Interleukin 28B during primary infection with cytomegalovirus.

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.

Arylacetamide deacetylase: a novel host factor with important roles in the lipolysis of cellular triacylglycerol stores, VLDL assembly and HCV production.

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.

Blood cytokine, chemokine and gene expression in cholestasis patients with intractable pruritis treated with a molecular adsorbent recirculating system: a case series.

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.

An analysis of regulatory T-cell and Th-17 cell dynamics during cytomegalovirus replication in solid organ transplant recipients.

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.

Clinical utility of cytomegalovirus cell-mediated immunity in transplant recipients with cytomegalovirus viremia.

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.

Clinical utility of molecular surveillance for cytomegalovirus after antiviral prophylaxis in high-risk solid organ transplant recipients.

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.

The clinical utility of whole blood versus plasma cytomegalovirus viral load assays for monitoring therapeutic response.

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.