A multi-host approach for the systematic analysis of virulence factors in Cryptococcus neoformans.

A multi-host approach was followed to screen a library of 1201 signature-tagged deletion strains of Cryptococcus neoformans mutants to identify previously unknown virulence factors. The primary screen was performed using a Caenorhabditis elegans-C. neoformans infection assay. The hits among these strains were reconfirmed as less virulent than the wild type in the insect Galleria mellonella-C. neoformans infection assay. After this 2-stage screen, and to prioritize hits, we performed serial evaluations of the selected strains, using the C. elegans model. All hit strains identified through these studies were validated in a murine model of systemic cryptococcosis. Twelve strains were identified through a stepwise screening assay. Among them, 4 (CSN1201, SRE1, RDI1, and YLR243W) were previously discovered, providing proof of principle for this approach, while the role of the remaining 8 genes (CKS101, CNC5600, YOL003C, CND1850, MLH3, HAP502, MSL5, and CNA2580) were not previously described in cryptococcal virulence. The multi-host approach is an efficient method of studying the pathogenesis of C. neoformans. We used diverse model hosts, C. elegans, G. mellonella, and mice, with physiological differences and identified 12 genes associated with mammalian infection. Our approach may be suitable for large pathogenesis screens.

The impact of antimicrobial resistance and aging in VAP outcomes: experience from a large tertiary care center.

BACKGROUND: Ventilator associated pneumonia (VAP) is a serious infection among patients in the intensive care unit (ICU). METHODS: We reviewed the medical charts of all patients admitted to the adult intensive care units of the Massachusetts General Hospital that went on to develop VAP during a five year period. RESULTS: 200 patients were included in the study of which 50 (25%) were infected with a multidrug resistant pathogen. Increased age, dialysis and late onset (≥ 5 days from admission) VAP were associated with increased incidence of resistance. Multidrug resistant bacteria (MDRB) isolation was associated with a significant increase in median length of ICU stay (19 vs. 16 days, p=0.02) and prolonged duration of mechanical ventilation (18 vs. 14 days, p=0.03), but did not impact overall mortality (HR 1.12, 95% CI 0.51-2.46, p=0.77). However, age (HR 1.04 95% CI 1.01-1.07, p=0.003) was an independent risk factor for mortality and age ≥ 65 years was associated with increased incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections (OR 2.83, 95% CI 1.27-6.32, p=0.01). CONCLUSIONS: MDRB-related VAP is associated with prolonged ICU stay and mechanical ventilation. Interestingly, age ≥ 65 years is associated with MRSA VAP.

Nocardiosis of the central nervous system: experience from a general hospital and review of 84 cases from the literature.

Central nervous system (CNS) nocardiosis is a rare disease entity caused by the filamentous bacteria Nocardia species. We present a case series of 5 patients from our hospital and a review of the cases of CNS nocardiosis reported in the literature from January 2000 to December 2011. Our results indicate that CNS nocardiosis can occur in both immunocompromised and immunocompetent individuals and can be the result of prior pulmonary infection or can exist on its own. The most common predisposing factors are corticosteroid use (54% of patients) and organ transplantation (25%). Presentation of the disease is widely variable, and available diagnostic tests are far from perfect, often leading to delayed detection and initiation of treatment. The optimal therapeutic approach is still undetermined and depends on speciation, but lower mortality and relapse rates have been reported with a combination of targeted antimicrobial treatment including trimethoprim/sulfomethoxazole (TMP-SMX) for more than 6 months and neurosurgical intervention.

Fusarium infection: report of 26 cases and review of 97 cases from the literature.

Fusarium species is a ubiquitous fungus that causes opportunistic infections. We present 26 cases of invasive fusariosis categorized according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria of fungal infections. All cases (20 proven and 6 probable) were treated from January 2000 until January 2010. We also review 97 cases reported since 2000. The most important risk factors for invasive fusariosis in our patients were compromised immune system, specifically lung transplantation (n = 6) and hematologic malignancies (n = 5), and burns (n = 7 patients with skin fusariosis), while the most commonly infected site was the skin in 11 of 26 patients. The mortality rates among our patients with disseminated, skin, and pulmonary fusariosis were 50%, 40%, and 37.5%, respectively. Fusarium solani was the most frequent species, isolated from 49% of literature cases. Blood cultures were positive in 82% of both current study and literature patients with disseminated fusariosis, while the remaining 16% had 2 noncontiguous sites of infection but negative blood cultures. Surgical removal of focal lesions was effective in both current study and literature cases. Skin lesions in immunocompromised patients should raise the suspicion for skin or disseminated fusariosis. The combination of medical monotherapy with voriconazole or amphotericin B and surgery in such cases is highly suggested.

Central nervous system aspergillosis: a series of 14 cases from a general hospital and review of 123 cases from the literature.

Central nervous system (CNS) aspergillosis is a highly fatal infection. We review the clinical presentation, diagnosis, and outcome of this infection and present a case series of 14 consecutive patients with CNS aspergillosis admitted to Massachusetts General Hospital (MGH) from 2000 to 2011. We also review 123 cases reported in the literature during that time. We included only proven CNS aspergillosis cases conforming to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal infections. In the MGH case series, neutropenia, hematologic malignancies, autoimmune diseases requiring steroid treatment, and solid organ transplantation were the predominant comorbid conditions. Notably, all MGH patients were immunosuppressed, and more than half (n = 8) had a history of previous brain injury, unrelated to their index hospitalization. For most MGH patients (11 of 14), the lung was the primary focus of aspergillosis, while 2 had paranasal sinus involvement, and 1 had primary Aspergillus discitis. Among reported cases, paranasal sinuses (27.6%) and the lung (26.8%) were the primary foci of infection, whereas 22% of those cases had no obvious primary organ involvement. Although a selection bias should be considered, especially among published cases, our findings suggest that patients who underwent neurosurgery had improved survival, with MGH and literature patients having 25% and 28.6% mortality, respectively, compared to 100% and 60.4%, respectively, among patients who received only medical treatment. Although this was not the case among MGH patients, CNS aspergillosis can affect patients without significant immune suppression, as indicated by the high number of reported immunocompetent cases. In conclusion, mortality among CNS aspergillosis patients remains high, and the infection may be more common among patients with previous brain pathology. When indicated, neurosurgical procedures may improve prognosis.

N-acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution.

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.

Update on the epidemiology and management of cryptococcal meningitis.

Despite recent improvements in the diagnosis and treatment of cryptococcosis, cryptococcal meningitis is responsible for > 600,000 deaths/year worldwide. The aim of this work is to provide an update on the developments in its epidemiology and management. Understanding the pathogenesis of Cryptococcus has improved, and new insights for the virulence of the fungus and the host response have enabled scientists to design new ways to confront this infection. Additionally, invertebrate model hosts have greatly facilitated the research in this field. Importantly, the epidemiology of Cryptococcus gattii has continued to evolve, and the emergence of this highly virulent species in immunocompetent populations, especially in Northwestern America and British Columbia, warrants increased awareness because delayed diagnosis and inappropriate antifungal therapy is associated with high mortality. Diagnosis remains a challenge, but new techniques for early and inexpensive identification of the pathogen are under development. Management can vary, based on the patient population (HIV-seropositive, organ transplant recipients or non-transplant/non-HIV). In most patients, amphotericin B with flucytosine continues to be the most appropriate induction therapy. However, in organ transplant recipients the use of liposomal amphotericin B improves mortality compared with deoxycholate amphotericin B. Also, the combination of amphotericin B with fluconazole seems to be a reasonable alternative, while fluconazole with flucytosine is superior to fluconazole monotherapy.

Polymerase chain reaction-based assays for the diagnosis of invasive fungal infections.

Currently accepted fungal diagnostic techniques, such as culture, biopsy, and serology, lack rapidity and efficiency. Newer diagnostic methods, such as polymerase chain reaction (PCR)-based assays, have the potential to improve fungal diagnostics in a faster, more sensitive, and specific manner. Preliminary data indicate that, when PCR-based fungal diagnostic assays guide antifungal therapy, they may lower patient mortality and decrease unnecessary antifungal treatment, improving treatment-associated costs and avoiding toxicity. Moreover, newer PCR techniques can identify antifungal resistance DNA loci, but the clinical correlation between those loci and clinical failure has to be studied further. In addition, future studies need to focus on the implementation of PCR techniques in clinical decision making and on combining them with other diagnostic tests. A consensus on the standardization of PCR techniques, along with validation from large prospective studies, is necessary to allow widespread adoption of these assays.

The effect of cumulative length of hospital stay on the antifungal resistance of Candida strains isolated from critically ill surgical patients.

Fluconazole is the first line of therapy for the management of candidiasis. However, fluconazole-resistant strains pose an emerging challenge in everyday clinical practice. In this study, we sought to determine whether cumulative length of hospital stay (CLOS) is a predictive factor for the acquisition of non-susceptible Candida strains to fluconazole. Thirty-three critically ill emergency surgery patients with 56 Candida isolates were enrolled in this prospective study. We divided our isolates according to their minimum inhibitory concentration (MIC) to fluconazole using 8 mcg/ml as a cutoff. We then compared the two groups with respect to basic demographics, antifungal agents prescribed, number of wide-spectrum antibiotics, duration of central venous catheter placement, elapsed time to positive culture, duration of prior hospital stay, and length of hospital stay. Non-susceptible fluconazole samples belonged to patients with a significantly longer prior hospital stay and a longer CLOS (P = 0.02 and 0.01, respectively). The difference between the 2 groups regarding non-albicans strains was statistically significant (P < 0.001). By fitting a non-parametric receiver-operating characteristics (ROC) curve into our analysis, a CLOS ≥ 29 days predicted the occurrence of non-susceptible strains with 90% sensitivity and 79.6% specificity (correct classification 81.5%). A CLOS ≥ 29 days is a strong predictor for the isolation of non-susceptible Candida isolates to fluconazole among critically ill emergency surgery patients. Clinicians should consider the duration of previous hospital stay when deciding on empiric antifungal therapy.

Statin therapy and decreased incidence of positive Candida cultures among patients with type 2 diabetes mellitus undergoing gastrointestinal surgery.

OBJECTIVE: To assess whether statin therapy decreases the incidence of cultures positive for Candida species among high-risk hospitalized patients with type 2 diabetes mellitus (DM). PATIENTS AND METHODS: We performed a retrospective cohort study analyzing the records of all patients with type 2 DM who were admitted to Massachusetts General Hospital for lower gastrointestinal tract surgery between January 1, 2001, and May 1, 2008. We defined statin exposure as the filling of at least 1 prescription of statins during the 6 months before hospitalization or during the current hospital stay. The primary outcome was a culture positive for Candida species during hospitalization. Clinical information on a wide range of covariates was collected. Logistic regression analysis was used to adjust for possible confounders. RESULTS: Of the 1019 patients who were eligible for the study, 493 (48%) were receiving statins. A total of 139 patients (14%) had at least 1 culture positive for Candida species during hospitalization. An adjusted multivariate model based on a backward stepwise elimination procedure showed that statin therapy significantly decreased the incidence of cultures positive for Candida species (odds ratio, 0.60; 95% confidence interval [CI], 0.38-0.96; P=.03) with a statistically significant prolonged time to event compared with no statin therapy (adjusted hazard ratio, 0.62; 95% CI, 0.44-0.88; P=.01). The benefit of statins was more prominent in patients with type 2 DM who had greater comorbidities (Charlson Comorbidity Index ≥2) (adjusted odds ratio, 0.47; 95% CI, 0.27-0.79; P=.01). CONCLUSION: Among patients with type 2 DM who underwent gastrointestinal surgery, use of statins correlated with a decreased incidence of cultures positive for Candida species.