Volume of fluids administered during resuscitation for severe sepsis and septic shock and the development of the acute respiratory distress syndrome.

PURPOSE: The purpose of this study was to examine the association between the volume of intravenous (IV) fluids administered in the resuscitative phase of severe sepsis and septic shock and the development of the acute respiratory distress syndrome (ARDS). MATERIALS AND METHODS: This was a retrospective cohort study of adult patients admitted with severe sepsis and septic shock at a large academic public hospital. The relationship between the volume of IV fluids administered and the development of ARDS was examined using multivariable logistic regression analysis. RESULTS: Among 296 patients hospitalized for severe sepsis and septic shock, 75 (25.3%) developed ARDS. After controlling for confounding variables, there was no significant association between the volume of IV fluids administered in the first 24 hours of hospitalization and the development of ARDS (odds ratio [OR], 1.05; 95% confidence interval [CI], 0.95-1.18). Serum albumin (OR, 0.52; 95% CI, 0.31-0.87) and Acute Physiology and Chronic Health Evaluation II score (OR, 1.08; 95% CI, 1.04-1.13) on admission were the most informative covariates for the development of ARDS in the regression model. CONCLUSIONS: For patients hospitalized for severe sepsis and septic shock, fluid administration to improve end-organ perfusion should remain the top priority in early resuscitation despite the potential risk of inducing ARDS.

Molecular epidemiology of carbapenem-nonsusceptible Acinetobacter baumannii in the United States.

Acinetobacter baumannii is emerging as an important nosocomial pathogen worldwide. We report molecular epidemiology of 65 carbapenem-nonsusceptible A. baumannii isolates identified from hospitals in New York, Pennsylvania, Florida, Missouri, Nevada, and California between 2008 and 2009. All isolates were subjected to pulsed-field gel electrophoresis (PFGE). Select isolates then underwent multilocus sequence typing (MLST). While the PFGE patterns tended to cluster within each hospital, sequence types (STs) belonging to the clonal complex 92 (CC92) and the pan-European clonal lineage II (EUII; worldwide clonal lineage 2) were predominant in all hospitals. Of them, ST122 and ST208 were the most common and were found in four of the six hospitals. Isolates belonging to the pan-European clonal lineages I and III were identified in one hospital each. Carbapenemase-encoding genes bla(OXA-23) and/or ISAba1-bla(OXA-51-like) were present among the majority of isolates. These findings suggest that carbapenem-nonsusceptible A. baumannii isolates found in U.S. hospitals constitute part of the global epidemic driven by CC92, but have unique STs other than ST92, which may be spreading by means of patient transfer between health care facilities within the United States.

Gene overexpression/suppression analysis of candidate virulence factors of Candida albicans.

We developed a conditional overexpression/suppression genetic strategy in Candida albicans to enable simultaneous testing of gain or loss of function in order to identify new virulence factors. The strategy involved insertion of a strong, tetracycline-regulated promoter in front of the gene of interest. To validate the strategy, a library of genes encoding glycosylphosphatidylinositol (GPI)-anchored surface proteins was screened for virulence phenotypes in vitro. During the screening, overexpression of IFF4 was found to increase the adherence of C. albicans to plastic and to human epithelial cells, but not endothelial cells. Consistent with the in vitro results, IFF4 overexpression modestly increased the tissue fungal burden during murine vaginal candidiasis. In addition to the in vitro screening tests, IFF4 overexpression was found to increase C. albicans susceptibility to neutrophil-mediated killing. Furthermore, IFF4 overexpression decreased the severity of hematogenously disseminated candidiasis in normal mice, but not in neutropenic mice, again consistent with the in vitro phenotype. Overexpression of 12 other GPI proteins did not affect normal GPI protein cell surface accumulation, demonstrating that the overexpression strategy did not affect the cell capacity for making such proteins. These data indicate that the same gene can increase or decrease candidal virulence in distinct models of infection, emphasizing the importance of studying virulence genes in different anatomical contexts. Finally, these data validate the use of a conditional overexpression/suppression genetic strategy to identify candidal virulence factors.

The iron chelator deferasirox protects mice from mucormycosis through iron starvation.

Mucormycosis causes mortality in at least 50% of cases despite current first-line therapies. Clinical and animal data indicate that the presence of elevated available serum iron predisposes the host to mucormycosis. Here we demonstrate that deferasirox, an iron chelator recently approved for use in humans by the US FDA, is a highly effective treatment for mucormycosis. Deferasirox effectively chelated iron from Rhizopus oryzae and demonstrated cidal activity in vitro against 28 of 29 clinical isolates of Mucorales at concentrations well below clinically achievable serum levels. When administered to diabetic ketoacidotic or neutropenic mice with mucormycosis, deferasirox significantly improved survival and decreased tissue fungal burden, with an efficacy similar to that of liposomal amphotericin B. Deferasirox treatment also enhanced the host inflammatory response to mucormycosis. Most importantly, deferasirox synergistically improved survival and reduced tissue fungal burden when combined with liposomal amphotericin B. These data support clinical investigation of adjunctive deferasirox therapy to improve the poor outcomes of mucormycosis with current therapy. As iron availability is integral to the pathogenesis of other infections (e.g., tuberculosis, malaria), broader investigation of deferasirox as an antiinfective treatment is warranted.

Optimization of a myeloid cell transfusion strategy for infected neutropenic hosts.

Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL-60. To further define the potential for HL-60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low-dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL-60 activation. In combination with activation, low-dose irradiation abrogated replication while sparing the candidacidal effects of the HL-60 cells. Infusion of irradiated, activated HL-60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL-60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.

Efficacy of the anti-Candida rAls3p-N or rAls1p-N vaccines against disseminated and mucosal candidiasis.

We have shown that vaccination with the recombinant N terminus of Als1p (rAls1p-N) protects mice against disseminated and oropharyngeal candidiasis. We now report that vaccination of mice with a related candidate, rAls3p-N, induces a broader antibody response than rAls1p-N and a similar cell-mediated immune response. The rAls3p-N vaccine was equally as effective as rAls1p-N against disseminated candidiasis but was more effective than rAls1p-N against oropharyngeal or vaginal candidiasis. Antibody titers did not correlate with protection against disseminated candidiasis, but delayed-type hypersensitivity did. The rAls3p-N vaccine is a promising new vaccine candidate for further exploration to prevent systemic and mucosal candidal infections.

The anti-Candida vaccine based on the recombinant N-terminal domain of Als1p is broadly active against disseminated candidiasis.

We have previously shown that vaccination with a vaccine based on the recombinant N-terminal domain of Als1p (rAls1p-N) protected BALB/c mice against disseminated infection caused by a single strain of Candida albicans (A. S. Ibrahim, B. J. Spellberg, V. Avenissian, Y. Fu, S. G. Filler, and J. E. Edwards, Jr., Infect. Immun. 73:999-1005, 2005, and B. J. Spellberg, A. S. Ibrahim, V. Avenissian, S. G. Filler, C. Myers, Y. Fu, and J. E. Edwards, Jr., Infect. Immun. 73:6191-6193, 2005). Here we show that the rAls1p-N vaccine also improves survival of outbred mice from disseminated candidiasis and that it is active against multiple virulent strains of C. albicans and non-C. albicans spp.

Current treatment strategies for disseminated candidiasis.

The incidence of disseminated candidiasis has increased dramatically over the past several decades. Fortunately, in recent years, a variety of new antifungal agents have become available to treat these infections. On the basis of efficacy, safety, and cost considerations, fluconazole is the agent of choice for the empirical treatment of disseminated candidiasis in nonneutropenic, hemodynamically stable patients, unless a patient is suspected to be infected with an azole-resistant species (i.e., Candida glabrata or Candida krusei). For hemodynamically unstable or neutropenic patients, agents with broader species coverage, such as polyenes, echinocandins, or, possibly, voriconazole, are preferred for empirical treatment of candidemia. Modification of the initial, empirical regimen depends on the response to therapy and the subsequent identification of the species of the offending pathogen. Echinocandins or high-dose polyenes are preferred for the treatment of infections with C. glabrata or C. krusei. Central venous catheters should be removed from all patients who have disseminated candidiasis, if feasible, and antifungal therapy should be administered to all patients who have candidemia or proven candidiasis.

The anti-Candida albicans vaccine composed of the recombinant N terminus of Als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice.

We have previously shown that intraperitoneal vaccination with the recombinant N terminus of Als1p (rAls1p-N) modestly improves survival during murine disseminated candidiasis. We now report marked efficacy with subcutaneous rAls1p-N vaccination. Efficacy is retained in neutropenic and corticosteroid-treated mice. The rAls1p-N vaccine is a promising candidate for the prevention of invasive candidiasis.

A phagocytic cell line markedly improves survival of infected neutropenic mice.

Disseminated candidiasis is a frequent infection in neutropenic patients, in whom it causes 50% mortality, despite antifungal therapy. As the duration of neutropenia is the strongest predictor of survival in neutropenic patients with invasive fungal infections, neutrophil transfusions are a logical, therapeutic option. However, significant technical barriers have prevented the clinical use of neutrophil transfusions. To overcome these barriers, we identified a human phagocytic cell line that could be administered to candidemic hosts in lieu of freshly harvested neutrophils. HL-60 cells killed Candida albicans in vitro. Activation of HL-60 cells with dimethyl sulfoxide and retinoic acid abrogated the cells' proliferation and augmented their killing of C. albicans. Administration of activated HL-60 cells to candidemic, neutropenic mice significantly improved survival (53% vs. 0%). Live HL-60 cells chemotaxed to sites of infection, phagocytized C. albicans, and reduced the fungal burden in key target organs. Although unactivated HL-60 cells also reduced tissue fungal burden in vivo, they did not improve survival as a result of their toxicity in infected mice. In contrast, no toxicity as a result of activated HL-60 cells was observed at up to 2 months of follow-up. To our knowledge, this is the first description of a cell line-based immunotherapy for an infectious disease. With further refinements, activated HL-60 cells have the potential to overcome the technical barriers to neutrophil transfusions.

Vaccination with recombinant N-terminal domain of Als1p improves survival during murine disseminated candidiasis by enhancing cell-mediated, not humoral, immunity.

Candida spp. are opportunistic fungal pathogens that are among the most common causes of nosocomial bloodstream infections. The mortality attributable to disseminated candidiasis is 40 to 50% despite antifungal therapy. Clearly, new strategies are needed to prevent this life-threatening infection. Because risk factors for disseminated candidiasis are well defined and frequently of limited duration, vaccination is an appealing prophylactic strategy. We have identified a cell surface protein, Als1p, that mediates adherence of Candida albicans to a variety of human substrates and plastic. Here we report that immunizing BALB/c mice with the recombinant N-terminal domain of Als1p (rAls1p-N) improved survival during a subsequent challenge with a lethal inoculum of C. albicans. The protective 20-mug dose of rAls1p-N significantly increased Candida stimulation of Th1 splenocytes and increased in vivo delayed-type hypersensitivity. In contrast, antibody titers did not correlate with protection. Finally, the vaccine was not protective in T-cell-deficient mice but was protective in B-cell-deficient mice. These data indicate that the mechanism of action of the rAls1p-N vaccine is stimulation of cell-mediated, rather than humoral, immunity against C. albicans. The majority of efforts to date have focused on the development of passive immunization strategies to prevent or treat disseminated candidiasis. In contrast, our results provide proof of principle for vaccination with an adhesin of C. albicans and emphasize the potential for cell-mediated immune modulation as a prophylactic or therapeutic strategy against disseminated candidiasis.