With increasing numbers of immune-compromised patients with malignancy, hematologic disease, and HIV, as well as those receiving immunosupressive drug regimens for the management of organ transplantation or autoimmune inflammatory conditions, the incidence of fungal infections has dramatically increased over recent years. Definitive diagnosis of pulmonary fungal infections has also been substantially assisted by the development of newer diagnostic methods and techniques, including the use of antigen detection, polymerase chain reaction, serologies, computed tomography and positron emission tomography scans, bronchoscopy, mediastinoscopy, and video-assisted thorascopic biopsy. At the same time, the introduction of new treatment modalities has significantly broadened options available to physicians who treat these conditions. While traditionally antifungal therapy was limited to the use of amphotericin B, flucytosine, and a handful of clinically available azole agents, current pharmacologic treatment options include potent new azole compounds with extended antifungal activity, lipid forms of amphotericin B, and newer antifungal drugs, including the echinocandins. In view of the changing treatment of pulmonary fungal infections, the American Thoracic Society convened a working group of experts in fungal infections to develop a concise clinical statement of current therapeutic options for those fungal infections of particular relevance to pulmonary and critical care practice. This document focuses on three primary areas of concern: the endemic mycoses, including histoplasmosis, sporotrichosis, blastomycosis, and coccidioidomycosis; fungal infections of special concern for immune-compromised and critically ill patients, including cryptococcosis, aspergillosis, candidiasis, and Pneumocystis pneumonia; and rare and emerging fungal infections.

Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection.

T cell-mediated immunity has been shown to play an important role in the host defense to Cryptococcus neoformans. Infections due to C. neoformans are increased in patients with AIDS who are deficient in the CD4+ subset of T lymphocytes. Thus, the effect of CD4+ (L3T4+) lymphocyte depletion on murine host defenses to C. neoformans was studied. The mAb GK 1.5 was administered to mice, and CD4+ T lymphocyte depletion was confirmed by the analysis of T cell subsets in blood, spleen, lymph node, and lung. Evidence of a functional defect was confirmed by demonstrating that the splenocytes of treated mice were unable to proliferate in response to class II incompatible spleen cells. Furthermore, delayed type hypersensitivity to C. neoformans was abrogated by CD4+ lymphocyte depletion. Mice depleted of CD4+ lymphocytes were inoculated with a virulent strain of C. neoformans by the i.v. or the intratracheal route. After i.v. inoculation of C. neoformans, the survival of mice depleted of CD4+ lymphocytes was reduced (27.8 +/- 1.8 vs 36.0 +/- 3.1 days, p less than 0.04). After intratracheal inoculation, C. neoformans disseminated from the lung to extrapulmonary organs. Dissemination occurred earlier in mice depleted of CD4+ lymphocytes compared to mice that received control antibody, and the burden of C. neoformans in extrapulmonary organs was greater in mice depleted of CD4+ lymphocytes than control mice. Surprisingly, there was no increase in the burden of C. neoformans in the lungs of CD4+ lymphocyte-depleted mice. Survival of mice inoculated with C. neoformans and depleted of CD4+ lymphocytes was reduced compared to control mice and was related to the increased rate of accumulation of organisms in the brains of treated mice. The mean survival of GK 1.5-treated mice was 34.1 +/- 0.9 days compared to control mice with a mean survival of 40.6 +/- 9 days (p less than 0.001). These data suggest that CD4+ lymphocytes play a prominent role in the host defense of infections due to C. neoformans, that CD4+ lymphocytes are required in extrapulmonary organs for optimal clearance of C. neoformans and that CD4+ lymphocytes are critical for survival of mice infected with C. neoformans.

Cryptococcus is a unique environmental fungus. Among the more than three dozen species of Cryptococcus, only C. neoformans and C. gattii commonly cause disease. Although many of these infections occur in immunocompromised patients, C. gattii has recently come to public attention because of an outbreak of devastating illness in immunocompetent individuals. The polysaccharide capsule of Cryptococcus is a major virulence factor, and in addition to surrounding the organism, it is also released into the environment. Cryptococcus is believed to enter the body through the lung causing pulmonary disease, but because of its neurotropic nature, the central nervous system is a major target organ. The major risk factors include HIV and organ transplantation. Depending on the site of infection and the patient's immune status, the clinical manifestations vary from asymptomatic to severe life-threatening disease. Treatment regimens depend on the immune status of the patient and the severity of the disease, and include both polyene and imidazole antifungal agents in addition to surgical adjuvant therapy. However, despite antifungal therapy, the mortality remains between 10 and 25% in patients with AIDS, and at least one-third of patients with cryptococcal meningitis experience mycological or clinical failure. Consequently, the mechanism of cryptococcal invasion, immune response, pathogenesis, and treatment continue to be areas of active study. With our advancing knowledge in these areas, we aim at better management for this devastating group of infections.

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl_2, or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.

Infection by Legionella pneumophila is believed to depend upon its ability to multiply within host alveolar macrophages. To investigate this, a site-specific mutation was introduced into a gene (mip) that encodes a 24,OOO-Da surface protein; an 80-fold loss of infectivity for both U937 cells and explanted human alveolar macrophages was observed. Further phenotypic analysis of the mutant strain has failed to show alterations in bacterial factors (e.g., proteinase, lipopolysaccharide) that have suspected roles in virulence. To substantiate that this mutation also results in reduced virulence in animals, the lethality and clinical illnesses produced by the parent and mutant L. pneumophila strains were compared in guinea pigs after intratracheal inoculation. The mutant strain produced fewer illnesses, slower-progressing disease, and fewer lethal infections than either the parent strain or a derivative of the mutant strain with the wild-type mip gene reintroduced. When sublethal inocula of the three strains were used, the mutant bacteria were recovered in slightly lower numbers from lung homogenates and in significantly lower numbers from the spleen, at 48 h, than were the other two test strains. Thus mip seems to be necessary for full virulence of L. pneumophila and may represent the first genetically defined virulence factor in this species.