In a eukaryotic kingdom of their own, fungi have interactions with humans ranging from colonization of skin by dermatophytes to local invasion of mucous membranes and deeper invasion with hematogenous dissemination. Various fungi are normal flora (e.g., Candida), opportunists that infect only persons with compromised host defenses (e.g., Aspergillus), and dimorphic organisms that are spore-forming molds in nature and convert into yeasts after entry as often occurs by inhalation (e.g., Histoplasma). Dimorphic fungi are capable of causing infections even in immunocompetent persons. The most frequently detected fungal structures in infected hosts are hyphae and budding yeast. Although detection of fungi invading tissue contributes to diagnosis, culture and mycologic identification provide a definitive answer.

Early diagnosis of invasive mucormycosis is important for timely therapeutic intervention, improved survival, and reduced morbidity. Given the importance of an accurate and rapid diagnosis of invasive mucormycosis to guide the timely initiation of amphotericin B and possible surgical intervention, a coordinated multidisciplinary approach of clinical assessment, diagnostic imaging, and laboratory assessment is necessary. Laboratory assessment for mucormycosis includes the conventional methods of direct examination and culture of tissue, respiratory secretions, bronchoalveolar lavage fluid, and other fluids. However, because conventional diagnostic tools are limited in their sensitivity, advanced molecular amplification systems, antigen detection assays, proteomic profiles, and metabolite detection may complement existing approaches to improve the rate of early diagnosis of invasive mucormycosis.

We designed, built, tested, space-qualified, launched, and collected telemetered data from low Earth orbit from PharmaSat, a 5.1-kg free flying "nanosatellite" that supported microbial growth in 48 microfluidic wells, dosed microbes with multiple concentrations of a pharmaceutical agent, and monitored microbial growth and metabolic activity using a dedicated 3-color optical absorbance system at each microwell. The PharmaSat nanosatellite comprised a structure approximately 10 x 10 x 35 cm, including triple-junction solar cells, bidirectional communications, power-generation and energy-storage system, and a sealed payload 1.2-L containment vessel that housed the biological organisms along with the fluidic, optical, thermal, sensor, and electronic subsystems. Growth curves for S. cerevisiae (Brewer's yeast) were obtained for multiple concentrations of the antifungal drug voriconazole in the microgravity conditions of low Earth orbit. Corresponding terrestrial control experiments were conducted for comparison.

Identification and classification of fungi from clinical samples are important for antifungal susceptibility testing and epidemiological investigation. Sequence-based molecular techniques are increasingly used in the identification and taxonomy characterization of fungal infections. Phenotypic characteristics used for the recognition and classification of fungi are those that are either easily observed or measured, or a combination of both. An ultimate goal of fungal classification is to draw inferred phylogenetic relationships. In brief, it is sequencing by synthesis, and it is based on the synthesis of cDNA from PCR amplicons. DNA targets that have been used for fungal identification and classification include rDNA, cytochrome b, β-tubulin, calmodulin, enolase, chitin synthase, heat shock protein, and other housekeeping and functional genes. The increased use of gene sequences to recognize different clades within traditional medically important species emphasizes the importance of using sequence data in contrast to species-specific probes to identify particular fungal species. Clinical laboratories are under increasing pressure to provide rapid identification and classification of fungal infections due to the growing number of immunocompromised patients that are susceptible to fungal infection, and the availability of targeted antifungal agents. Sequencing and database comparison of PCR amplicons coupled with phylogenetic methods provide a robust strategy for species recognition, especially for uncommon and emerging pathogenic fungi.

Human pythiosis, caused primarily by the aquatic oomycete, Pythium insidiosum, is an emerging but uncommon infection in North America. The infection is frequently life-threatening and is often initially unrecognized due to its rarity and similar presentation to certain fungal infections.We report a case of skin and soft tissue pythiosis in a patient without significant underlying comorbidities acquired in a New Mexico hot spring and review its successful treatment. We also review all reported pythiosis cases in North America.Eleven confirmed cases of human pythiosis acquired in North America were identified. The majority of cases occurred in children (64%), ten of eleven cases were acquired in the southern U.S., Mexico, Central America or the Caribbean and four of the eleven individuals succumbed to the infection.With recognition and aggressive surgical and medical treatment good clinical outcomes can be achieved when treating human pythiosis.

Fungi are eukaryotic, unicellular or multicellular organisms that are larger and genomically more complex than bacteria. The fungal cell wall is complex and has polysaccharides, proteins, sugars, and glycoproteins. Plasma membranes of fungi contain ergosterol, which is the primary target for antifungal drugs such as amphotericin B. Although more than 1.3 million fungal species exist in the environment, only about 150 are pathogenic to humans.1 The virulence factors of fungi resemble those of bacteria, such as possession of a capsule, adhesion molecules, toxins, free radicals, and so forth. Thus, fungi can elicit acute exudative, necrotizing, and granulomatous reactions in tissues. Although some generalizations are possible, the diverse structural and antigenic properties of individual fungi produce unique patterns of infection in individual hosts.2,5

The incidence of cerebral phaeohyphomycosis, an infection caused by a dark-pigmented fungus, is increasing. The infection may mimic a high-grade glioma clinically and radiographically. Magnetic resonance spectroscopy may be helpful in differentiating the two. We report two cases to increase the awareness of cerebral phaeohyphomycosis in the clinical neurosciences. Early biopsy establishing the diagnosis, followed by aggressive combined surgical and medical management is necessary for a good outcome.

A 57-year-old male presented with dermatosis of the dorsum of the foot consisting of tumefaction, deformity and sinus tract formation. The direct examination of exudates as well as the biopsy tissue, demonstrated the presence of black granules. A dematiaceous fungus was isolated from the lesions and was identified by ribosomal DNA sequencing as Cladophialophora bantiana. This is the second report of this fungus as an etiologic agent of eumycetoma in humans. Clinical and mycologic cure was achieved after 20 months of treatment with itraconazole at a starting dose of 300 mg/day that was tapered during the course of therapy. The patient's isolate had an itraconazole MIC of 0.012 microg/ml.

Fungal infections can be diagnosed on the basis of mycologic, immunologic, clinical, and histopathologic information. Of these procedures, histopathology can provide important diagnostic information in a relatively short period of time, but is limited in that much of the information obtained from the examination of tissue sections can provide only tentative fungal identification, unless specialized techniques such as immunofluorescence are used or when the etiologic agent has distinctly unique structures (such as spherules containing endospores). These limitations are particularly evident in the case of filamentous fungi, as many of the opportunistic filamentous pathogens have similar tissue morphology (Fig. 3.1). Although this often makes identification of these organisms from tissue sections essentially impossible, examination of tissue sections is critical in determining whether fungi are involved in invasion, colonization, or simply have been recovered in culture as contaminants. The technique is rapid, inexpensive, and accurate, and the information it provides can have enormous and immediate patient care implications. Even though the most specific method in identifying a fungus is to recover it in culture so that it can be properly and accurately identified, cultures may not be submitted for various reasons. In addition, some organisms, such as Lacazia loboi (Fig. 3.2) and Rhinosporidium seeberi (Fig. 3.3), have not yet been grown in vitro. In the absence of culture recovery, the diagnosis of fungal infection via histology rests on the size, morphology, and staining properties of fungal elements in tissue along with the assessment of invasion of normally sterile tissue and body fluids. 2. STAINS

The incidence of invasive fungal infections has increased dramatically over the past two decades, mostly due to an increase in the number of immunocompromised patients.1–4 Patients who undergo chemotherapy for a variety of diseases, patients with organ transplants, and patients with the acquired immune deficiency syndrome have contributed most to the increase in fungal infections.5 The actual incidence of invasive fungal infections in transplant patients ranges from 15% to 25% in bone marrow transplant recipients to 5% to 42% in solid organ transplant recipients.6,7 The most frequently encountered are Aspergillus species, followed by Cryptococcus and Candida species. Fungal infections are also associated with a higher mortality than either bacterial or viral infections in these patient populations. This is because of the limited number of available therapies, dose-limiting toxicities of the antifungal drugs, fewer symptoms due to lack of inflammatory response, and the lack of sensitive tests to aid in the diagnosis of invasive fungal infections.1 A study of patients with fungal infections admitted to a university-affiliated hospital indicated that community-acquired infections are becoming a serious problem; 67% of the 140 patients had community-acquired fungal pneumonia.8

The incidence of invasive fungal infections has increased dramatically over the past two decades, mostly due to an increase in the number of immunocompromised patients. l -4 Patients who undergo chemotherapy for a variety of diseases, patients with organ transplants, and patients with the acquired immune deficiency syndrome have contributed most to the increase in fungal infections.s The actual incidence of invasive fungal infections in transplant patients ranges from 15% to 25% in bone marrow transplant recipients to 5% to 42% in solid organ transplant recipients.,7 The most frequently encountered are Aspergillus species, followed by Cryptococcus and Candida species. Fungal infections are also associated with a higher mortality than either bacterial or viral infections in these patient popUlations. This is because of the limited number of available therapies, dose-limiting toxicities of the antifungal drugs, fewer symptoms due to lack of inflammatory response, and the lack of sensitive tests to aid in the diagnosis of invasive fungal infections. I A study of patients with fungal infections admitted to a university-affiliated hospital indicated that communityacquired infections are becoming a serious problem; 67% of the 140 patients had community-acquired fungal pneumonia. There is also an increase in nosocomial fungal infections.9 Fungi are eukaryotic, unicellular to multicellular organisms that have chitinous cell walls, and reproduce asexually, sexually, or both ways. Fungal cells are larger and genomically more complex than bacteria. Their cell wall contains polysaccharides, proteins, and sugars, and their antigens are rich in complex polysaccharides and glycoproteins. Plasma membranes of fungi contain ergosterol, which is the primary target for antifungals such as amphotericin B. Although there are more than 1.3 million fungal species in the environment, only about 150 are known to be pathogenic to humans. For detailed taxonomy of the fungi, several texts are available. W-12 The virulence factors of fungi resemble those of bacteria, such as possession of a capsule, adhesion molecules, toxins, free radicals, etc. Thus, fungi can elicit similar tissue reactions as bacteria, including acute exudative, necrotizing, and granulomatous reactions. Pathogenicity of fungi depends on the virulence of the particular fungus, the infecting dose, the route of infection, and the immune status of the host. Other factors that influence the pathogenicity include the coexistence of other infections in the host, the organs affected, and other underlying diseases. There is no clear-cut evidence that fungal infections are contagious, with the exception of dermatophytoses, pityriasis versicolor, and candidiasis of the newborn. However, accidental inoculation or direct contamination of an open wound can result in transmission of the etiologic agent of infection.13 Hence care should be taken to avoid direct contact when handling contaminated bodily discharges and tissues. Most pulmonary infections begin in the lungs following inhalation of aerosolized fungi from the environment. ,14 Once the fungi reach the lungs, the infection can remain localized, or it can disseminate to produce severe systemic disease that is often fatal. Rarely, the gastrointestinal tract and skin may be the primary focus of systemic infection with secondary involvement of the lungs, especially in immunocompromised patients. The discussion of fungi in this chapter is confined to invasive pulmonary infections. Fungi that cause invasive pulmonary infection can be divided in two main groups: (1) primary or true pathogens, and (2) opportunistic pathogens. The primary or true pathogenic fungi (also referred to as endemic fungi) infect healthy, immunologically competent individuals.,16 These fungi can be very aggressive, and produce severe disseminated and fatal infections in immunocompromised patients. While endemic fungi such as Histoplasma capsulatum and Coccidioides immitis are the most common, a number of other fungi have emerged as important, though less common pathogens. Penicillium marneffei, Fusarium species, Scedosporium species, and Malassezia species are increasing in incidence as opportunistic infections in immunocompromised hosts, especially in those with

A case of cerebral aspergillosis was diagnosed by the detection of Aspergillus flavus-specific DNA in brain biopsy and serum specimens. The diagnosis was also supported by detection of elevated levels of galactomannan and (1-->3)-beta-d-glucan in serum specimens. Despite the presence of dichotomously branched septate hyphae in brain biopsy, the culture remained negative. The inability to isolate the organism in culture suggested that combined therapy of AmBisome and caspofungin was fungicidal for the fungus in the brain abscess.