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Preservation techniques that maintain the viability of an organ graft between retrieval from the donor and implantation into the recipient remain a critical aspect of solid organ transplantation. While traditionally preservation is accomplished with static cold storage, advances in ex vivo dynamic machine perfusion, both hypothermic and normothermic, have allowed for prolongation of organ viability and recovery of marginal organs effectively increasing the usable donor pool. However, the use of these novel machine perfusion technologies likely exposes the recipient to additional infectious risk either through clonal expansion of pathogens derived during organ recovery or de novo exogenous acquisition of pathogens while the organ remains on the machine perfusion circuit. There is a paucity of high-quality studies that have attempted to quantify infection risk, although it appears that prolonging the time on the machine perfusion circuit and normothermic parameters increases the risk of infection. Conversely, the use of ex vivo machine perfusion unlocks new opportunities to detect and treat donor-derived infections before implantation into the recipient. This review seeks to reveal how the use of ex vivo machine perfusion strategies may augment the risk of infection in the organ recipient as well as outline ways that this technology could be leveraged to enhance our ability to manage donor-derived infections., (© 2024 Wiley Periodicals LLC.)

Background: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is a clinical syndrome with various causes. It is not uncommon that COPD patients presenting with dyspnea have multiple causes for their symptoms including AECOPD, pneumonia, or congestive heart failure occurring concurrently., Methods: To identify clinical, radiographic, and laboratory characteristics that might help distinguish AECOPD from another dominant disease in patients with a history of COPD, we conducted a retrospective cohort study of hospitalized patients with admitting diagnosis of AECOPD who were screened for a prospective randomized controlled trial from Sep 2016 to Mar 2018. Clinical characteristics, course in hospital, and final diagnosis at discharge were reviewed and adjudicated by two authors. The final diagnosis of each patient was determined based on the synthesis of all presenting signs and symptoms, imaging, and laboratory results. We adhered to AECOPD diagnosis definitions based on the GOLD guidelines. Univariate and multivariate analyses were performed to identify any associated features of AECOPD with and without other acute processes contributing to dyspnea., Results: Three hundred fifteen hospitalized patients with admitting diagnosis of AECOPD were included. Mean age was 72.5 (SD 10.6) years. Two thirds (65.4%) had spirometry defined COPD. The most common presenting symptom was dyspnea (96.5%), followed by cough (67.9%), and increased sputum (57.5%). One hundred and eighty (57.1%) had a final diagnosis of AECOPD alone whereas 87 (27.6%) had AECOPD with other conditions and 48 (15.2%) did not have AECOPD after adjudication. Increased sputum purulence (OR 3.35, 95%CI 1.68-6.69) and elevated venous pCO2 (OR 1.04, 95%CI 1.01 - 1.07) were associated with a diagnosis of AECOPD but these were not associated with AECOPD alone without concomitant conditions. Radiographic evidence of pleural effusion (OR 0.26, 95%CI 0.12 - 0.58) was negatively associated with AECOPD with or without other conditions while radiographic evidence of pulmonary edema (OR 0.31; 95%CI 0.11 - 0.91) and lobar pneumonia (OR 0.13, 95%CI 0.07 - 0.25) suggested against the diagnosis of AECOPD alone., Conclusion: The study highlighted the complexity and difficulty of AECOPD diagnosis. A more specific clinical tool to diagnose AECOPD is needed., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Competing Interests: Intérêts concurrents: Aucun déclaré.

Competing Interests: Competing interests: None declared.

Acanthamoeba spp. are the causative pathogens of several infections, including amoebic keratitis (AK), a vision-threatening infection. Acanthamoebae from corneal specimens of patients with AK harbor bacterial endosymbionts, which may increase virulence. We sought to understand the spectrum of bacterial endosymbionts present in clinical isolates of Acanthamoeba spp. identified in our reference parasitology laboratory. Isolates of Acanthamoeba spp. obtained from our biobank of anonymized corneal scrapings were screened for potential endosymbionts by PCR using primer pairs detecting bacteria belonging to orders Chlamydiales, Rickettsiales, or Legionellales and pan16S primers. Three primer pairs specific to the 18s rRNA gene of Acanthamoeba spp. were used for the amplification of Acanthamoeba DNA used for sequencing. Sanger sequencing of all PCR products was performed, followed by BLAST analysis for species identification. We screened 26 clinical isolates of Acanthamoeba spp. for potential endosymbionts. Five isolates (19%) were found to contain bacterial DNA belonging to Legionellales. Three (11%) contained members of the Rickettsiales and Pseudomonas genticulata was detected in a Rickettsia -positive sample. One strain (4%) contained Neochlamydia hartmannellae , a member of the Chlamydiales order. Bacterial endosymbionts are prevalent in clinical strains of Acanthamoeba causing AK isolated from corneal scrapings. The demonstration of these organisms in clinical Acanthamoeba isolates supports a potential exploration of anti-endosymbiont therapeutics as an adjuvant therapy in the treatment of AK.

Backgound: Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity., Methods: Expression of proinflammatory biomarkers, including interleukin (IL) 1β, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species., Results: LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1β (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status., Conclusions: Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Suboptimal agreement between molecular assays for the detection of Acanthamoeba spp. in clinical specimens has been demonstrated, and poor assay sensitivity directly imperils the vision of those affected by amoebic keratitis (AK) through delayed diagnosis. We sought to develop and validate a single Taqman real time PCR assay targeting the Acanthamoeba 18S rRNA gene that could be used to enhance sensitivity and specificity when paired with reference assays., Methods: Biobanked DNA from surplus delinked AK clinical specimens and 10 ATCC strains of Acanthamoeba was extracted. Sequence alignment of 66 18S rRNA regions from 12 species of Acanthamoeba known to cause keratitis informed design of a new TaqMan primer set. Performance of the new assay was compared to the 2 assays used currently in our laboratory., Results: Among 24 Acanthamoeba-positive and 83 negative specimens by the CDC reference standard, performance characteristics of the newly designed primer set were as follows: sensitivity 100%, specificity 94%, PPV 82.8%, and NPV 100%. Compared to culture, sensitivity of the new primer set was 100%, and specificity 96%. No cross-reactivity of the primer set to non-acanthamoebae, including Balamuthia and Naegleria, was found., Conclusions: We have validated a real time PCR assay for the diagnosis of AK, and in doing so, have overcome important barriers to rapid and sensitive detection of acanthamoebae, including limited sensitivity and specificity of commonly used assays.

Background: Amoebic keratitis is a potentially blinding eye infection caused by ubiquitous, free-living, environmental acanthamoebae, which are known to harbor bacterial endosymbionts. A Chlamydia-like endosymbiont has previously enhanced Acanthamoeba virulence in vitro. We investigated the potential effect of Acanthamoeba-endosymbiont coinfection in a human corneal tissue model representing clinical amoebic keratitis infection., Methods: Environmental and corneal Acanthamoeba isolates from the American Type Culture Collection were screened for endosymbionts by amplifying and sequencing bacterial 16S as well as Chlamydiales-specific DNA. Each Acanthamoeba isolate was used to infect EpiCorneal cells, a 3-dimensional human corneal tissue model. EpiCorneal cells were then treated with azithromycin, doxycycline, or control medium to determine whether antibiotics targeting common classes of bacterial endosymbionts attenuated Acanthamoeba virulence, as indicated by decreased observed cytopathic effect and inflammatory biomarker production., Results: A novel endosymbiont closely related to Mycobacterium spp. was identified in Acanthamoeba polyphaga 50495. Infection of EpiCorneal cells with Acanthamoeba castellanii 50493 and A. polyphaga 50372 led to increased production of inflammatory cytokines and cytopathic effects visible under microscopy. These increases were attenuated by azithromycin and doxycycline., Conclusions: Our findings suggest that azithromycin and doxycycline may be effective adjuvants to standard antiacanthamoebal chemotherapy by potentially abrogating virulence-enhancing properties of bacterial endosymbionts., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Background and Objectives: The establishment at our center of a dedicated regional anesthesia service in 2008-2009 has resulted in a marked increase in single-shot brachial plexus blocks (sBPBs) for ambulatory wrist fracture surgery. Despite the documented benefits of regional over general anesthesia (GA), there has been a perceived increase among sBPB patients in postoperative return rates for pain at our institution. We conducted a retrospective quality improvement project to examine this., Methods: After exemption from human ethics board review, we sought to identify and contact all wrist fracture surgery patients treated at our center between 2003 and 2012. Our primary outcome was the incidence of unplanned physician visits (office/clinic or emergency department) for pain in the first 48 hours after surgery. Other main outcomes included the incidence of seeking any form of medical attention for pain and self-reporting of severe pain in the first 48 hours., Results: Of 1008 identified patients, 419 could be contacted; 195 qualified for analysis. The incidence of unplanned physician visits in the first 48 hours was 12% (13 of 118) among sBPB patients versus 4% (3 of 77) in GA patients (odds ratio [OR], 3.1; 95% confidence interval [95% CI], 0.8-11.1; P = 0.11). More sBPB versus GA patients sought any form of medical attention for pain (20% vs 5%; OR, 4.7; 95% CI, 1.4-10.9; P = 0.003). Similarly, more sBPB patients reported severe postoperative pain (41% vs 10%; OR, 5.9; 95% CI, 2.6-13.4; P < 0.0001)., Conclusions: Patients who received sBPBs for ambulatory wrist fracture surgery had a higher rate of unplanned health care resource utilization caused by pain after hospital discharge than those undergoing GA. These findings warrant confirmation in a prospective trial and emphasize the need for a defined postdischarge analgesic pathway as well as the potential merits of perineural home catheters.

mexCD-oprJ is an envelope stress-inducible multidrug efflux operon of Pseudomonas aeruginosa. A gene encoding a homologue of the NfxB repressor of this operon, PA4596, occurs downstream of oprJ and was proposed as a second repressor of this efflux operon. Inactivation of this gene had no impact on mexCD-oprJ expression in cells not exposed to envelope stress although its loss under envelope stress conditions yielded a > 10-fold increase in mexCD-oprJ expression. Consistent with PA4596 functioning as a mexCD-oprJ repressor, the purified protein was able to bind to a DNA fragment carrying the mexCD-oprJ promoter region. Expression of PA4596 was induced under conditions of envelope stress dependent on the AlgU envelope stress sigma factor, consistent with PA4596 operating under envelope stress conditions where it possibly serves to moderate envelope stress-inducible mexCD-oprJ expression. nfxB mutants showed elevated PA4596 expression and purified NfxB bound to DNA encompassing the PA4596 upstream region, an indication that NfxB functions as a repressor of PA4596 expression. Elimination of PA4596 in P. aeruginosa lacking nfxB and hyperexpressing mexCD-oprJ had no additional impact on mexCD-oprJ expression, regardless of the presence of envelope stress, suggesting that PA4596 repressor activity may be dependent on NfxB. This envelope stress-regulated repressor of mexCD-oprJ has been renamed esrC., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The mexCD-oprJ multidrug efflux operon of Pseudomonas aeruginosa is regulated by the NfxB repressor. Two forms of NfxB have been reported [Shiba et al. (1995). J Bacteriol 177, 5872) although mutagenesis studies here confirm that the larger protein (199 amino acids, 22.4 kDa) is the functional repressor. NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding. Two-hybrid assays confirmed that NfxB is a multimer, with the C-terminal two-thirds of the repressor required for multimerization. Random mutagenesis identified several mutations within the C-terminal region of NfxB required for multimerization, all of which mapped to a three-helix subdomain of the C-terminal region in a structural model of the repressor, which may thus represent the multimerization domain. These mutations compromised NfxB binding to its target DNA in electromobility shift assays, and their introduction into the chromosome of P. aeruginosa enhanced mexCD-oprJ expression and promoted multidrug resistance, consistent with the functional NfxB repressor being a multimer. Site-directed and spontaneous nfxB mutants showing increased mexCD-oprJ expression and multidrug resistance were also recovered, with mutations mapping to the three-helix subdomain again impacting multimerization and DNA binding. Mutations mapping to the N-terminal helix-turn-helix motif implicated in DNA binding did not impact multimerization although they did render the repressor insoluble and unsuitable for mobility shift assays. Size exclusion column chromatography demonstrated that wild-type NfxB forms tetramers in solution, although a mutant form of the repressor carrying a G192D substitution near the C terminus of the protein and compromised for DNA binding and repressor activity forms dimers. These results suggest that NfxB operates as a tetramer (dimer of dimers) and that the C terminus of the protein serves as a tetramerization domain.