The vast majority of fungi have evolved to reside in various environmental niches where they grow readily on vicinal organic substrates and are protected from deleterious conditions. Although these exogenous fungi are unable to penetrate the intact surfaces of healthy hosts, they may be acquired accidentally by traumatic exposure to resident fungi in soil, water, air, or vegetation. Once fungal cells have breached the cutaneous or mucosal surfaces, such as the skin, or the respiratory, urinary, or gastrointestinal tract, they are repelled by innate host defenses. Potentially pathogenic fungi must be able to grow at 37°C, acquire essential nutrients from the host, and evade the immune responses. The few hundred environmental fungi with these attributes represent only a tiny percentage of global species. Unfortunately, a few highly prevalent fungi with these abilities are able to cause opportunistic, invasive infections in patients with compromised host defenses (eg, aspergillosis and cryptococcosis). Overall, the most prevalent mycoses are caused by noninvasive molds, the dermatophytes, which have adapted to grow on the skin, hair, or nails, and by endogenous species of Candida and Malassezia, which are members of the human mycobiome. However, regardless of their source, with the exception of dermatophytes, pathogenic fungi are not contagious, and transmission among humans or animals is extremely rare. This chapter describes the most prevalent mycoses, but new pathogens are reported every year. There is also brief coverage of two different mechanisms whereby fungi may cause human disease—ingestion of fungal toxins or exposure to fungal cell wall components that elicit IgE-mediated allergic responses.

In general, the most definitive methods to establish the diagnosis of a fungal infection are culture of the pathogen, microscopic examination, detection of species-specific fungal DNA, and serology. Since these methods vary in their avail ability, specificity, sensitivity, methodology, and turnaround time, it is prudent to utilize several diagnostic strategies.

A. Specimens

 Clinical specimens collected for microscopy and culture are determined by the site(s) of infection and the condition of the patient. All specimens should be obtained using aseptic technique, especially with specimens from normally sterile sites, such as blood, tissue biopsies, and cerebrospinal fluid. Specimens from nonsterile body sites include skin and subcutaneous lesions, nasopharyngeal or genital swabs, sputa, urine, and wounds. To minimize bacterial growth, specimens should be transported to the diagnostic laboratory within 2 hours. Whenever a fungal infection is suspected, alert the diagnostic laboratory because special stains and culture media have been developed for the detection of pathogenic fungi.

B. Microscopic Examination

One or two drops of an aqueous or serous specimen, such as sputum, urine, spinal fluid, or aspirate, can be placed on a glass slide in a drop of 10–20% potassium hydroxide (KOH), and after adding a coverslip, the slide is examined under the microscope with the low- and high-power (450×) objectives. KOH dissolves any tissue cells, and the resistant, highly refractory fungal cell walls become more visible. This procedure can also be used to examine skin scrapings or minced tissue samples. The sensitivity of the KOH solution is improved by the addition of calcofluor white, which is a nonspecific fungal cell wall stain that is visible with a fluorescent microscope. The detection of fungi in pus, viscous exudates, and minced tissue can also be examined with KOH preps by gently heating the slide to dissolve excess tissue debris and inflammatory cells. Fungi can also be observed in blood smears, CSF, and other preps treated with Gram or Wright stain.

In formalin-fixed biopsy specimens, fungi can be detected with the routine hematoxylin and eosin (H&E) histopathological stain. However, specialized fungal cell wall stains are more sensitive. The two most common stains are Gomori methenamine silver (GMS) and periodic acid Schiff (PAS), which stain fungal walls black or red, respectively. Other specialized stains, such as capsule stains for Cryptococcus, are described in subsequent sections.

Although the sensitivity of microscopic examinations varies with the mycosis and the extent of disease, this examination can be performed very quickly and is often definitive. In the host, most fungi grow as yeasts, hyphae, or a combination of yeasts and pseudohyphae. Table 1 lists the spectrum of in vivo fungal structures. In many cases, the pathogens that are present only as yeasts are sufficiently distinctive in size and shape to establish an immediate diagnosis. In other cases, based on fungal appearance (eg, nonseptate hyphae or brownish cell walls) and the specimen site (eg, superficial or systemic), the list of possible fungal agents is considerably narrowed.

Table1. Key Fungal Structures Observed in Microscopic Examinations of Clinical Specimens

C. Culture

In most cases, the culture is more sensitive than the direct examination, and a portion of the material collected for microscopy should be cultured. The traditional mycological medium, Sabouraud’s dextrose agar (SDA), contains glucose and modified peptone (pH 7.0), supports the growth of fungi, and restricts the growth of bacteria. The morphologic characteristics of fungi used for identification have been described from growth on SDA. However, other media, such as inhibitory mold agar (IMA), enhance the recovery of fungi from clinical specimens. To culture medical fungi from nonsterile specimens, antibacterial antibiotics (eg, gentamicin and chloramphenicol) and cycloheximide are added to the media to inhibit bacteria and saprobic molds, respectively. After cultures are obtained, Potato Dextrose Agar stimulates the production of conidia.

For culturing blood, several commercial broth media have been developed for bacteria and/or fungi. Most yeast species in blood can be detected and subcultured from these media within 3 days. However, molds may require several weeks of incubation to become positive, and hence, special procedures must be used to optimize their recovery. Yeasts grow better at 37°C and molds at 30°C. When a dimorphic fungus is suspected, multiple media and incubation temperatures are recommended. The ideal method for likely cases of fungemia is a commercial lysis-centrifugation (Isolator®) tube to which blood is directly added. The tube contains an anticoagulant and detergent to lyse the blood cells, releasing any phagocytized fungal cells, and the tube is then centrifuged to pellet any fungi. After decanting the supernatant fluid, the pellet is suspended, streaked on agar plates of mycologic media, and incubated. Positive cultures of most yeasts or molds can be identified by morphological and physiological phenotypes. Several commercial microculture systems for yeasts are able to generate substrate assimilation profiles, and these profiles can be compared with large databases to identify most pathogenic species of yeasts. As described later, specialized media are available to assist in the rapid identification of Candida species (eg, CHROMagar®).

D. Serology

The following sections of this chapter will explain how the detection of specific antibodies or antigens in serum or CSF can provide useful diagnostic and/or prognostic information. In immunocompetent patients, positive antibody tests may confirm the diagnosis, and negative tests may exclude fungal disease. However, the interpretation of each serologic test depends upon its sensitivity, specificity, and positive or negative predictive value in the population of patients being tested.

E. Molecular Methods

 An increasing number of clinical laboratories have implemented methods based on the detection of fungal nucleic acids, proteins, or antigens to identify pathogenic fungi in clinical specimens or after their recovery in culture. Multiple approaches have been published, and in-house as well as commercial systems are available, but none have been widely adopted. In the next decade one or more of these methods may become routine, especially if they can be automated, provide rapid throughput, and detect multiple microbes. In addition, some platforms have the potential for point-of-care usage. Most DNA-based methods use PCR to amplify fungus-specific sequences of ribosomal DNA or other conserved genes. By comparing the DNA sequences of a clinical isolate with databases of thousands of fungal DNA sequences, the genus or species of an unknown fungus can be identified. This approach has been used to identify cultures of hundreds of fungal taxa. In addition, a variety of reports have used PCR to detect fungal DNA (mostly Candida or Aspergillus) in blood and other specimens.

Automated commercial instruments for the DNA-based identification of pathogenic fungi have been developed by several companies, including Luminex Molecular Diagnos tics®, Gen-Probe®, LightCycler® SeptiFast, and MicroSeq®. For detection, they typically use oligonucleotide probes that emit a signal amplified by fluorescence, chemical reagents, or enzyme immunoassay. For detecting fungal cells in slide preparations, a PNA-FISH® (peptide-nucleic acid fluorescent in situ hybridization) test kit with species-specific probes can be used.

DNA-based fingerprinting methods, such as multilocus sequence typing (MLST), have identified phylogenetic sub populations of many pathogenic fungi, including species of Candida, Cryptococcus, Aspergillus, and Coccidioides. Some of these molecular subgroups are clinically relevant as they are associated with differences in geographic distribution, susceptibility to antifungal drugs, clinical manifestations, or virulence. Some of these subgroups may represent cryptic species that cannot be differentiated by phenotypic methods.

Another molecular method that is gaining currency because of its application to pathogenic bacteria as well as fungi involves the extraction of microbial proteins, which are then submitted to mass spectroscopy. Pathogens are identified by comparing their protein spectral patterns with those in databases of previously tested species. With the ease of sample preparation and the commercial availability of automated instruments, this method—matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS)—is becoming more accessible. In several studies, MALDI-TOF-MS has proven to be more accurate and faster than conventional culture methods. Most of the initial reports focused on the identification of species of Candida, but the databases have since expanded to include hundreds of other fungal species.

Similar to the rapid test for endotoxin, the clotting cascade of the horseshoe crab (Limulus) hemolymph is also triggered by fungal cell wall β-(1,3)-d-glucan (Fungitell®). This polysaccharide is shed during infection, and its coagulation of hemolymph has been exploited to quantify its concentration in blood and spinal fluid. β-(1,3)-d-glucan levels of ≥80 pg/mL are positive and associated with invasive candidiasis, aspergillosis, dimorphic pathogens, and other mycoses.

F. Antifungal Susceptibility Testing

After the diagnosis of a systemic mycosis, appropriate antifungal chemotherapy is initiated. As discussed at the end of this chapter, there are three major classes of antifungal drugs. However, many pathogenic fungi are capable of devel oping resistance to antifungal drugs, and the clinical micro biology laboratory is often required to assess in vitro the susceptibility (or resistance) of the patient’s fungal isolate versus a specific antifungal drug. The Clinical Laboratory Standards Institute has developed protocols for in vitro testing the minimal inhibitory concentration (MIC) of pathogenic fungal isolates against approved drugs. For example, a clinical yeast isolate can be cultured in broth, suspended at a standard concentration of colony-forming units (CFU) per milliliter, and placed in microtiter wells containing a range of concentrations of a specific antifungal; after incubation for 24 or 48 hours, the lowest concentration of drug (µg/mL) to inhibit growth is the MIC. Since most antifungal drugs are fungistatic rather than fungicidal, the MIC is a helpful guide to effective treatment. For many fungus-antifungal drug combinations, MIC breakpoints have been developed to designate drug concentrations as susceptible (S), intermediate (I or S-DD, susceptible dose-dependent), or resistant (R). However, a drug’s in vitro effectiveness does not always correlate with its efficacy in the patient. There are alterna tive approved methods and commercial kits for measuring the MIC, such as the Etest® (bioMérieux) and Sensititre YeastOne® (Trek Diagnostics).

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متى يصبح محيط الخصر "مؤشر خطر"؟

دراسة تكشف دور "العوامل الوراثية" في تحديد عمر الإنسان

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المزيد

الآخبار التكنلوجية

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المزيد

مواضيع ذات صلة

Cutaneous mycoses

General Properties, Virulence, and Classification of Pathogenic Fungi

Conventional Yeast identification methods

Commercially available yeast identification systems

Approach to identification of the Yeasts

Specimen collection, transport, and processing of The Yeasts

Cryptococcus neoformans: pathogenesis and spectrum of disease

Candida albicans: pathogenesis and spectrum of disease

General characteristics of the Yeasts

Laboratory diagnosis of Pneumocystis jiroveci

Pathogenesis and spectrum of disease of Pneumocystis jiroveci

General Characteristics of Pneumocystis jiroveci