Molecular epidemiology of Clostridioides difficile isolates in a non-outbreak setting at a comprehensive cancer center.

Ribotyping was performed on Clostridioides difficile isolates from patients with malignancies. Thirty-one (27.9%) isolates from 111 episodes of colitis were recovered representing 14 ribotypes with 25 (80.6%) belonging to 6 ribotypes (014/020, 1/VPI/077/087, 05/015, 015/046, 05/053, 106/174). We identified three novel ribotypes with 1 carrying gene encoding for binary toxin.

Low-Level Cytomegalovirus Antigenemia Promotes Protective Cytomegalovirus Antigen-Specific T Cells after Allogeneic Hematopoietic Cell Transplantation.

Previous studies have reported a beneficial effect from cytomegalovirus (CMV) reactivation after allogeneic hematopoietic stem cell transplantation (alloHCT) on immune reconstitution. We determined the CMV antigenemia level associated with increased CMV antigen-specific T cells (CASTs) at day +100 and decreased CMV reactivation after day +100. CMV reactivation and CASTs were measured with CMV antigenemia and CMV-specific major histocompatibility complex multimers. The analysis consisted of 775 CAST measurements obtained before and 30, 100, and 365 days post-alloHCT from 327 consecutive patients treated between 2008 and 2016. Detectable CASTs correlated with recipient (P < .0001) and donor (P < .0001) CMV seropositivity pre-alloHCT. CMV reactivation before day +100 was associated with a higher proportion of patients who achieved ≥3 CASTs/µL by day +100 (61% with versus 39% without reactivation, P < .001). In alloHCT recipients at high risk for CMV reactivation (R+D±) with a maximum of grade II acute graft-versus-host-disease, reactivating CMV before day +100 and achieving ≥3 versus <3 CASTs/µL at day +100 was associated with reduced CMV reactivation from day +100 to +365 (27% versus 62%, P = .04). This protective effect was observed with low-level but not high-level CMV reactivation (<5 versus ≥5/50,000 polymorphonuclear leukocytes + pp65, respectively). These findings suggest low-level CMV reactivation may be beneficial and that treatment may be delayed until progression. These findings will need validation in prospective clinical trials using CMV PCR and antigenemia assays.

Discontinuation of Systematic Surveillance and Contact Precautions for Vancomycin-Resistant Enterococcus (VRE) and Its Impact on the Incidence of VRE faecium Bacteremia in Patients with Hematologic Malignancies.

OBJECTIVE To study the effect of discontinuation of systematic surveillance for vancomycin-resistant Enterococcus (VRE) and contact isolation of colonized patients on the incidence of VRE bacteremia SETTING A hematology-oncology unit with high prevalence of VRE colonization characterized by predominantly sporadic molecular epidemiology PARTICIPANTS Inpatients with hematologic malignancies and recipients of hematopoietic stem cell transplantation METHODS The incidence of VRE bacteremia was measured prospectively during 2 different 3-year time periods; the first during active VRE surveillance and contact precautions and the second after discontinuation of these policies. We assessed the collateral impact of this policy change on the incidence of bacteremia due to methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile infection even though we maintained contact precautions for these organisms. Incidence of infectious events was measured as number of events per 1,000 patients days per month. Time series analysis was used to evaluate trends. RESULTS The incidence of VRE bacteremia remained stable after discontinuation of VRE surveillance and contact precautions. The incidence of MRSA bacteremia and Clostridium difficile infection for which we continued contact precautions also remained stable. Aggregated antibiotic utilization and nursing hours per patient days were similar between the 2 study periods. CONCLUSION Active surveillance and contact precautions for VRE colonization did not appear to prevent VRE bacteremia in patients with hematologic malignancies and recipients of hematopoietic stem cell transplantation with high prevalence of VRE characterized by predominantly sporadic molecular epidemiology.

Correlation between Circulating Fungal Biomarkers and Clinical Outcome in Invasive Aspergillosis.

Objective means are needed to predict and assess clinical response in patients treated for invasive aspergillosis (IA). We examined whether early changes in serum galactomannan (GM) and/or ß-D-glucan (BDG) can predict clinical outcomes. Patients with proven or probable IA were prospectively enrolled, and serial GM and BDG levels and GM optical density indices (GMI) were calculated twice weekly for 6 weeks following initiation of standard-of-care antifungal therapy. Changes in these biomarkers during the first 2 and 6 weeks of treatment were analyzed for associations with clinical response and survival at weeks 6 and 12. Among 47 patients with IA, 53.2% (25/47) and 65.9% (27/41) had clinical response by weeks 6 and 12, respectively. Changes in biomarkers during the first 2 weeks were associated with clinical response at 6 weeks (GMI, P = 0.03) and 12 weeks (GM+BDG composite, P = 0.05; GM, P = 0.04; GMI, P = 0.02). Changes in biomarkers during the first 6 weeks were also associated with clinical response at 6 weeks (GM, P = 0.05; GMI, P = 0.03) and 12 weeks (BDG+GM, P = 0.02; GM, P = 0.02; GMI, P = 0.01). Overall survival rates at 6 weeks and 12 weeks were 87.2% (41/47) and 79.1% (34/43), respectively. Decreasing biomarkers in the first 2 weeks were associated with survival at 6 weeks (BDG+GM, P = 0.03; BDG, P = 0.01; GM, P = 0.03) and at 12 weeks (BDG+GM, P = 0.01; BDG, P = 0.03; GM, P = 0.01; GMI, P = 0.007). Similar correlations occurred for biomarkers measured over 6 weeks. Patients with negative baseline GMI and/or persistently negative GMI during the first 2 weeks were more likely to have CR and survival. These results suggest that changes of biomarkers may be informative to predict and/or assess response to therapy and survival in patients treated for IA.

NADPH oxidase promotes neutrophil extracellular trap formation in pulmonary aspergillosis.

NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.

Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice.

Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ß-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.

NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury.

Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury.

Acyclovir prophylaxis against varicella zoster virus reactivation in multiple myeloma patients treated with bortezomib-based therapies: a retrospective analysis of 100 patients.

BACKGROUND: Previous studies have indicated that, in patients with multiple myeloma (MM), bortezomib is associated with an increased incidence of herpes zoster, resulting from reactivation of latent varicella zoster virus (VZV). OBJECTIVE: Our objective was to determine whether increased risk of VZV reactivation could be abrogated by using prophylactic acyclovir. METHODS: We retrospectively evaluated 100 consecutive MM patients treated with bortezomib-based therapies at the Roswell Park Cancer Institute for development of herpes zoster. Frontline and relapsed/refractory patients were included, and patients received bortezomib alone or in combination with agents such as doxorubicin, melphalan, or dexamethasone. All patients received >4 weeks of acyclovir prophylaxis (400 mg twice daily), which was initiated prior to starting treatment with bortezomib and discontinued 4 weeks following bortezomib. RESULTS: Median patient age was 62 years, 57% were male, and most (56%) had Durie-Salmon stage IIIA MM. None of the 100 MM patients receiving acyclovir prophylaxis developed herpes zoster during treatment with bortezomib, irrespective of patients receiving a wide variety of concomitant antimyeloma therapies and regardless of response to bortezomib-based therapy. One additional patient, found to be noncompliant with acyclovir therapy, experienced VZV reactivation, having received 3 cycles of bortezomib (3 weeks each cycle) in combination with cyclophosphamide and dexamethasone. LIMITATIONS: Limitations of the study include its small size and retrospective nature. CONCLUSIONS: The increased risk of VZV reactivation observed in previous studies of bortezomib-based therapy was completely abrogated in this series of patients who received prophylaxis with acyclovir.

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense.

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.

Role of NADPH oxidase in host defense against aspergillosis.

NADPH oxidase plays a critical role in antimicrobial host defense, as evident in chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal diseases. Invasive aspergillosis and other moulds are the major cause of mortality in CGD. We also learn from CGD patients that NADPH oxidase plays an important role in regulating inflammation; CGD patients are prone to developing inflammatory diseases such as inflammatory bowel disease, obstructive granulomata of the genitourinary tract, and hypersensitivity pneumonitis. Indeed, the NADPH oxidase plays an essential role in calibrating innate and T-cell responses to control the growth of inhaled fungi while protecting against excessive and injurious inflammation. Knowledge gained on the mechanisms by which NADPH oxidase kills fungi and regulates inflammation may lead to new therapeutics for CGD and will have broad relevance to understanding host-pathogen interactions between mammals and ubiquitous moulds to which we are continually exposed.

Antifungal prophylaxis and therapy in patients with hematological malignancies and hematopoietic stem cell transplant recipients.

Patients with acute leukemia and hematopoietic stem cell transplant recipients are at risk of a spectrum of invasive fungal diseases corresponding to the type and intensity of immunosuppression. The development of newer antifungal agents has broadened therapeutic options. In the 1990s, lipid formulations of amphotericin B became widely used as safer alternatives to amphotericin B deoxycholate. In addition, fluconazole was shown to be beneficial as a yeast-active prophylaxis in hematopoietic stem cell transplant recipients. In the past decade, the antifungal armamentarium was further enhanced with the availability of extended-spectrum azoles and echinocandins. The development of effective broad-spectrum antifungal agents has led to their use as prophylaxis rather than delaying treatment until clinical signs of infection manifest. Antigen-based and PCR-based diagnostic adjuncts facilitate earlier detection of invasive fungal diseases compared with conventional culture, and have been incorporated into strategies in which initiation or modification of an antifungal regimen is targeted to patients with the highest likelihood of having fungal disease. Here, we review the pharmacological data and major clinical trials that guide the use of antifungals, as well as areas of uncertainty and future perspectives.

Infection control measures to prevent invasive mould diseases in hematopoietic stem cell transplant recipients.

Invasive mould diseases, particularly aspergillosis, are important causes of morbidity and mortality in allogeneic stem cell transplant recipients. Mould spores are ubiquitous in the environment. Guidelines established by the Centers for Disease Control (CDC) and other authoritative organizations focus on approaches to reduce exposure to mould spores. These recommendations include avoidance of areas and activities expected to result in high levels of mould spores (e.g., construction, gardening) and use of specially designed units (protected environments) where additional standards (e.g., HEPA-filtered rooms) are in place to minimize mould exposure. These recommendations are based on consensus criteria and limited clinical data largely derived from single-center retrospective studies. In addition, highly immunocompromised stem cell transplant recipients are commonly managed as outpatients, where engineering standards of the inpatient protected environment are not feasible. In the absence of an outbreak with an identified environmental source (e.g., a contaminated air vent), it is not possible to reliably distinguish community-acquired from nosocomial aspergillosis. Adherence to infection control guidelines, acknowledging their limitations, combined with evidence-based targeted antifungal prophylaxis for the highest risk transplant recipients, is likely to be the most effective approach to prevent invasive mould diseases.

Prevention and treatment of invasive fungal diseases in neutropenic patients.

PURPOSE OF REVIEW: Despite advances in the diagnosis and management, invasive fungal diseases contribute substantially to the morbidity and mortality of patients with prolonged neutropenia. RECENT FINDINGS: Major advances in the prevention, diagnosis, and treatment of invasive fungal diseases have occurred with the introduction of fungal markers and new antifungal agents over the past decade. The newer broad-spectrum azoles and echinocandins, due to their acceptable safety profiles and efficacy, have emerged as valuable options as antifungal prophylaxis and therapy. Empirical antifungal therapy, a strategy in which persistent neutropenic fever triggers the addition of an antifungal agent, remains a common practice. However, the development of fungal diagnostic markers (e.g. galactomannan and beta-glucan assays) combined with radiologic imaging has given potential to more targeted preemptive antifungal strategies. SUMMARY: With the validation of preemptive strategies in clinical trials, we will likely have evidence-based targeted use of antifungals. PCR-based techniques are also promising tools for preemptive strategies and are undergoing evaluation. Knowledge of host genetic factors may be important in stratifying the risk for fungal disease during periods of high risk.

Antifungal agents in hematopoietic stem cell transplantation.

Invasive fungal infections are major complications of stem cell transplantation associated with significant morbidity and mortality. Allogeneic stem cell transplant recipients are at a significantly greater risk for fungal infection than recipients of autologous transplantation. Although with the wide use of fluconazole prophylaxis the incidence and associated mortality of invasive candidiasis has been minimized, mold diseases remain a significant complication during periods of prolonged immunosuppression for graft versus host disease. Posaconazole prophylaxis during periods of high risk was recently demonstrated to be effective in preventing fungal infections and associated mortality. Preemptive strategy employing laboratory markers and serial CT scans to identify mold infection at an early stage is promising. However its efficacy has to be validated in clinical trials. Several new antifungal agents have been introduced lately, characterized by improved safety profile and broader antifungal spectrum. Voriconazole has become the standard of care for the treatment of invasive aspergillosis. Finally there has been increasing interest on combination therapy for invasive aspergillosis due to the high rate of failure of the currently available antifungals, especially in the profoundly immunocompromised host.

Defining responses to therapy and study outcomes in clinical trials of invasive fungal diseases: Mycoses Study Group and European Organization for Research and Treatment of Cancer consensus criteria.

Invasive fungal diseases (IFDs) have become major causes of morbidity and mortality among highly immunocompromised patients. Authoritative consensus criteria to diagnose IFD have been useful in establishing eligibility criteria for antifungal trials. There is an important need for generation of consensus definitions of outcomes of IFD that will form a standard for evaluating treatment success and failure in clinical trials. Therefore, an expert international panel consisting of the Mycoses Study Group and the European Organization for Research and Treatment of Cancer was convened to propose guidelines for assessing treatment responses in clinical trials of IFDs and for defining study outcomes. Major fungal diseases that are discussed include invasive disease due to Candida species, Aspergillus species and other molds, Cryptococcus neoformans, Histoplasma capsulatum, and Coccidioides immitis. We also discuss potential pitfalls in assessing outcome, such as conflicting clinical, radiological, and/or mycological data and gaps in knowledge.

Broad-spectrum antifungal prophylaxis in patients with cancer at high risk for invasive mold infections: point.

Invasive fungal infections (IFIs) are a leading cause of infection-related mortality in patients with acute leukemia and prolonged neutropenia and in allogeneic hematopoietic stem cell transplant (HSCT) recipients with graft-versus-host disease (GVHD). Although invasive candidiasis was the principal IFI predating fluconazole prophylaxis, invasive aspergillosis and other mold infections now cause most deaths from fungal infection in this patient population. The availability of broad-spectrum antifungal agents that can be safely administered over prolonged periods has stimulated interest in using mold-active prophylactic agents early as prophylaxis rather than later as therapy for suspected or documented IFIs. Two recent, prospective, randomized trials have shown a clear benefit of posaconazole prophylaxis in patients with myelodysplastic syndrome and acute mye-logenous leukemia with prolonged neutropenia and in allogeneic HSCT recipients with severe GVHD. In contrast, the peer-reviewed published database on the strategy of preemptive antifungal therapy, in which yeast-active prophylaxis (fluconazole) or no antifungal prophylaxis is used initially and modifications are triggered by a combination of laboratory markers and chest CT scans, is currently limited to an open-label feasibility study. Does sufficient evidence currently exist that the net benefit of the preemptive approach is at least on a par with posaconazole prophylaxis in the specific patient groups that were studied? The authors believe not and that more research is needed before the preemptive strategy can be recommended.

Ganciclovir inhibits lymphocyte proliferation by impairing DNA synthesis.

Cytomegalovirus (CMV) disease-related mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients has dramatically declined because of ganciclovir prophylaxis and preemptive therapeutic strategies. However, ganciclovir has not improved overall survival in randomized studies despite effectively preventing overt CMV disease. Moreover, recurrent posttransplant CMV antigenemia, associated with prolonged ganciclovir exposure, is a predictor of increased relapse of malignancy. We examined the hypothesis that ganciclovir itself may have a negative impact on immune reconstitution by testing the effect of ganciclovir on normal human lymphocytes in vitro. T-lymphocyte activation and proliferation, as measured by PHA-induced (3)H-thymidine uptake, was greatly reduced at therapeutic concentrations of ganciclovir (10 microg/mL) but not for foscarnet (300 microM/L). Moreover, ganciclovir impaired bromodeoxyuridine incorporation in proliferating lymphocytes, but did not impair lymphocyte survival or induce lymphocyte apoptosis. Collectively, these results show that ganciclovir suppresses T-lymphocyte proliferation in vitro by inhibiting DNA synthesis; with implications for T-lymphocyte function following allogeneic BMT.

In vitro susceptibilities of 217 clinical isolates of zygomycetes to conventional and new antifungal agents.

We evaluated the in vitro susceptibilities of 217 zygomycetes to amphotericin B, ketoconazole, fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, and flucytosine. The significant in vitro activity of posaconazole against several species appears to support its reported clinical efficacy. Decreased susceptibility to amphotericin B was noted with Cunninghamella bertholletiae.