Diagnosis

Diagnosis of the Mycoses

The diagnosis of mycoses is based on clinical observation and laboratory examination of clinical specimens. Diagnosis of superficial mycoses affecting the skin and mucous membranes, such as candidiasis, is often straightforward. The characteristic appearance of the lesions frequently suggests the diagnosis. Moreover, specimens from the site of infection are easily obtained for laboratory isolation, examination, and identification [Richardson and Warnock, 1997].

In contrast, diagnosis of deep-tissue mycotic infections, such as invasive candidiasis or aspergillosis, presents significant challenges. The manifestations of infection can vary, depending on the site of the infection and the host's immune status. Moreover, clinical signs and symptoms are often nonspecific and difficult to distinguish from those seen with bacterial infections. Commonly, a fever that persists or develops while the patient is receiving a broad-spectrum antibiotic is the first indication of fungal infection. Collection of clinical specimens to isolate and identify fungal pathogens may be difficult, especially when the focus of infection is unknown. Moreover, the results of serologic tests for systemic fungal pathogens can be difficult to interpret and may be unreliable [Richardson and Warnock, 1997; Sugar and Layman, 1997].

With systemic mycoses, a definitive diagnosis may not be made until late in the course of the infection or at autopsy. Because a delay in treatment can be fatal, antifungal therapy is commonly initiated empirically. The decision to treat is based on clinical suspicion, which takes into account the patient's clinical status, the failure of antibiotic therapy, and the presence of known predisposing factors for fungal infection [Richardson and Warnock, 1997].

Clinical Features

Oropharyngeal or Esophageal Candidiasis

Oropharyngeal candidiasis is a fungal infection of the mucosal surfaces of the mouth and pharynx caused by various species of the genus Candida. Esophageal candidiasis involves the mucous membrane of the esophagus.

Risk factors

Both local and systemic factors can predispose a patient to candidiasis. Neonates are at risk because of the immaturity of their immune systems [Epstein and Polsky, 1998]. They can acquire oropharyngeal candidiasis through birth from a mother with vaginal candidiasis or through exposure to infected bottle nipples or the skin of an infected nurse or the mother [Epstein and Polsky, 1998].

Diabetics are at increased risk for oropharyngeal candidiasis. Although the mechanism for risk in diabetics has not been clearly established, elevated glucose levels and a reduced chemotactic factor in the saliva, altered neutrophil function, and reduced saliva volume may be the cause. Patients with impaired salivary gland function are at increased risk for oropharyngeal candidiasis because saliva secretion causes a dilutional effect, its movement clears organisms from mucosal surfaces, and it also contains antimicrobial proteins. Denture wearers also have an increased susceptibility to oropharyngeal candidiasis due to enhanced adherence of Candida to acrylic, reduced saliva flow under dentures, improperly fitted dentures, or poor oral hygiene.

Use of a broad-spectrum antibiotic increases the risk of developing oropharyngeal candidiasis because the drug alters the normal oral flora and creates a favorable environment for Candida organisms to proliferate. Long-term use of a corticosteroid inhaler can increase the risk of oropharyngeal candidiasis, possibly by suppressing cellular immunity and inhibiting phagocytosis.

Disruption of the oral mucosa, whether due to mucositis or decreased mucosal regeneration caused by chemotherapy, puts patients at a higher risk for oropharyngeal candidiasis. Cancer chemotherapy can cause transient xerostomia, which can disrupt the oral mucosa and interfere with the bacterial flora of the mouth, allowing overgrowth of Candida and inducing acute oropharyngeal candidiasis. Oropharyngeal candidiasis has been reported in up to 33% of leukemia patients and is commonly a marker for previously unrecognized esophageal candidiasis in those with cancer. Samonis et al. [1998] evaluated 22 patients with various neoplasms and oral thrush and found that 21 of these patients also had endoscopically and microbiologically proven esophageal candidiasis, but only 10 of these patients had symptoms of esophageal candidiasis.

Patients with human immunodeficiency virus (HIV) infection are at high risk for oropharyngeal candidiasis, which is present in up to 95% of patients with acquired immunodeficiency syndrome (AIDS) because their CD4 lymphocytes are depleted [Epstein and Polsky, 1998]. Furthermore, its presence is a predictor of disease progression in these patients [Epstein and Polsky, 1998]. Oropharyngeal candidiasis is estimated to have a positive predictive value of about 90% for esophageal candidiasis in HIV-infected patients, making the presence of oropharyngeal candidiasis a moderately useful diagnostic marker for esophageal candidiasis [Darouiche, 1998].

Signs and symptoms

Symptoms associated with oropharyngeal candidiasis include burning, increased sensitivity, altered taste, change in the sense of smell, dysphagia, and odynophagia [Epstein and Polsky, 1998]. Oropharyngeal candidiasis manifests in several forms. The "classic" form is pseudomembranous candidiasis, or thrush, which is characterized by soft, yellowish white plaques on the oral mucosa that can be removed with vigorous rubbing, leaving red or bleeding sites.

Erythematous oropharyngeal candidiasis involves the dorsal aspect of the tongue and the palate and appears as red mucosa with loss of papillae on the tongue and patchy, red changes in the palate. The palate becomes painful and erythematous with few, if any, white patches. In denture stomatitis, the palatal mucosa in contact with the dentures becomes affected and is chronically, but asymptomatically, erythematous and edematous, and a hyperplastic response can occur. Angular cheilitis is an inflammatory reaction caused mainly by C. albicans at the corners of the mouth, characterized by painful, red fissures. Leukoplakia due to hyperplastic candidiasis appears as bilateral, elevated, white mucosal lesions on the buccal mucosa, tongue, lips, and the bottom of the mouth [Epstein and Polsky, 1998].

In esophageal candidiasis, the gross appearance of the esophageal mucosa is graded from 0 (normal appearance) to 4 (includes confluent, linear, nodular elevated plaques with superficial ulceration and narrowing of the esophageal lumen) [Samonis et al., 1998]. In a study of esophageal candidiasis in AIDS patients, most plaques appeared yellow to tan, although some had a black-green appearance. As the severity of disease progressed, the scattered plaques coalesced, circumferentially coating the mucosal surface. Under the plaques, the mucosa was inflamed, but without true ulceration in most patients. Grade 4 plaque extended into the esophageal lumen. In most patients, the disease was more prominent in the proximal esophagus. Ulceration was present in 32% of patients; however, ulceration could be attributed to Candida in only four of these patients [Wilcox and Schwartz, 1996].

Many patients with esophageal candidiasis are asymptomatic [Darouiche, 1998]. In a study of 31 HIV-negative patients, Ortuno Cortes et al. [1997] found that the most common signs and symptoms of esophageal candidiasis were dysphagia-odynophagia (35%); epigastric, xiphoid, or retrosternal pain (16%); and hiccups (3.2%). Comorbid conditions in patients with esophageal candidiasis included chronic obstructive pulmonary disease, diabetes mellitus, cirrhosis, digestive tract bleeding, other esophageal, duodenal or gastric lesions, and stroke.

Medical tests

Candidiasis can be diagnosed by examining smears from the lesions using Gram stain or potassium hydroxide [Epstein and Polsky, 1998]. Because Candida organisms are normal commensals of the gastrointestinal (GI) tract, diagnosis cannot be confirmed solely by detection of Candida. A characteristic clinical lesion and response to antifungal treatment are necessary to confirm the diagnosis [Epstein and Polsky, 1998].

To diagnose oropharyngeal candidiasis, smears of the oral lesions are examined microscopically for yeasts and hyphae or pseudohyphae [Samonis et al., 1998]. The diagnostic procedure of choice for esophageal candidiasis is endoscopy with biopsy [Ortuno Cortes et al., 1997]. Although cultures of the mycotic exudate can determine the type of fungus that caused the infection, they do not distinguish between pathogenic and commensal fungi. Serologic testing has not proven reliable as a diagnostic tool [Ortuno Cortes et al., 1997].

Invasive Candidiasis

Invasive candidiasis is a systemic fungal disease caused by various species of the genus Candida. Candidemia is defined as the isolation of pathogenic species of Candida from one or more blood specimens [Rodriguez et al., 1997; Vincent et al., 1998] and includes syndromes ranging from catheter-related candidemia in a nonneutropenic patient to acute disseminated candidiasis in a profoundly neutropenic host [Rodriguez et al., 1997]. Disseminated candidiasis refers to Candida infections in multiple, noncontiguous organs caused by hematogenous spread of the pathogen [Vincent et al., 1998]. Hepatosplenic candidiasis (chronic disseminated candidasis) is a specific syndrome seen in patients with leukemia or bone marrow transplantation who have experienced prolonged severe neutropenia [Rodriguez et al., 1997].

Risk factors

Common risk factors noted among patients with candidemia are the presence of a central venous catheter (85%), use of parenteral hyperalimentation (61%), prior (especially multiple) antibiotic use (94%), and neutropenia (29%) [Grohskopf and Andriole, 1997]. Neutrophils are one of the body's primary defenses against systemic Candida infection; therefore, neutropenic patients or patients with impaired phagocytic function are susceptible to invasive Candida infections [Wingard, 1995]. Other factors that predispose patients to invasive Candida infections involve breaching of the integument, impaired phagocytic function [Grohskopf and Andriole, 1997], prolonged hospital stays, use of steroids, colonization with Candida [Rodriguez et al., 1997], severe underlying disease (hematologic malignancies, organ transplants, diabetes, chronic renal failure), intravenous drug use, trauma or burns, or abdominal or thoracic surgery [Grohskopf and Andriole, 1997].

The major endogenous reservoir of Candida is the GI tract [Pfaller, 1996]. Treatments for underlying disease can cause significant damage to the GI tract, allowing the colonizing Candida organisms easier access to the bloodstream, resulting in hematogenous spread [Wingard, 1995]. For example, when leukemic patients become neutropenic, they usually are treated with a combination of antimicrobial agents that may alter the bowel flora and promote proliferation of Candida organisms [Jarvis, 1995]. Chemotherapy simultaneously denudes the bowel allowing systemic spread of Candida [Jarvis, 1995]. Additional evidence of endogenous sources of infection is the isolation of patient-unique strains of Candida from multiple anatomic sites over time, which are usually of the same DNA type [Pfaller, 1996].

Candidiasis may also be acquired from exogenous sources. Pfaller reported a high prevalence of hand carriage of Candida among hospital personnel [Pfaller, 1996]. Outbreaks of Candida infection have been reported in high-risk patients due to transmission from contaminated intravenous solutions and devices, food services, and hyperalimentation solutions [Pfaller, 1996].

Signs and symptoms

Patients with Candida infections present with a wide variety of symptoms due, in part, to the wide variety of infections, ranging from catheter-related candidemia in nonneutropenic patients to acute disseminated candidiasis in profoundly neutropenic hosts. In catheter-related candidemia, the abrupt appearance of fever in a critically ill medical or surgical patient prompts blood cultures that may or may not yield Candida species. Usually no localized signs are present, and the infection appears limited to the bloodstream [Rodriguez et al., 1997].

Patients with acute disseminated candidiasis are critically ill with overwhelming Candida-related sepsis [Rodriguez et al., 1997]. Signs and symptoms are similar to those of bacteremia and include fever, leukocytosis, hypotension, and possibly septic shock. Hematogenous spread of the organism causes dissemination to multiple organs causing diffuse microabcesses. The most commonly affected sites are the skin, eye, kidney and brain, but the myocardium, heart valves, joints, and bone may also be involved [Grohskopf and Andriole, 1997]. Development of purpura fulminans and hemorrhagic bullae has been described in patients with Candida sepsis. Other consequences of the hematogenous spread of Candida are suppurative peripheral thrombophlebitis, Candida-associated diarrhea, and Candida epiglottitis [Wright and Wenzel, 1997].

Chronic disseminated candidiasis (hepatosplenic candidiasis) occurs primarily in cancer patients who are recovering from neutropenia. These patients have right upper quadrant abdominal pain and are persistently febrile after recovery from neutropenia, despite the use of broad-spectrum antibiotics [Grohskopf and Andriole, 1997].

Aspergillosis

Aspergillosis is a spectrum of fungal diseases caused by members of the genus. Aspergillus molds are ubiquitous, occurring in plant material and soil, and have been isolated from foods and indoor air [Medical Mycology Research Center, University of Texas Medical Branch, 1999]. The clinical manifestation and severity of the disease depends on the immunologic state of the patient and the species of Aspergillus involved. Factors such as underlying debilitating disease (AIDS, tuberculosis, cancer), neutropenia, disruption of normal flora, and therapy with corticosteroids or antibiotics can predispose the patient to colonization, invasive disease, or both [Medical Mycology Research Center, University of Texas Medical Branch, 1999].

Risk factors

Important predisposing factors for invasive aspergillosis are neutropenia (<1000 cells/mm³), use of corticosteroids and use of broad-spectrum antibiotics [Mylonakis et al., 1998]. Patients who have undergone bone marrow transplants for treatment of acute hematologic malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, and lymphoma) [Denning et al., 1998] are at particular risk because of their neutropenic status or treatment with corticosteroids for graft versus host disease [Ribaud et al., 1999]. Others at risk for aspergillosis are patients who have received solid organ transplants, especially lung transplant [Yeldandi et al., 1995].

Signs and symptoms

Many patients with suspected invasive pulmonary aspergillosis present with fever refractory to broad-spectrum antibiotics, cough, low-grade chest pain, dyspnea (more common in diffuse disease) and occasionally hemoptysis, which can be life-threatening [Denning, 1998]. Some 25-33% of patients initially have no symptoms attributable to invasive pulmonary aspergillosis. Patients with cerebral aspergillosis may show fever, headache, hemiparesis, or other nonspecific findings (such as seizures and alterations in mental status) [Denning, 1998; Pagano et al., 1996].

Medical tests

Although diagnosis of aspergillosis can be difficult, its early detection and treatment are important factors in improving the clinical outcome and survival rate of patients with invasive aspergillosis. The mortality rate for untreated invasive aspergillosis is nearly 100%. Ideally the diagnosis should be based on biopsy, which would histologically prove the presence of Aspergillus hyphae. However, many patients are not stable enough to undergo such an invasive procedure [Denning, 1998].

Patients may show pulmonary infiltrates on chest X-rays. Thoracic computerized tomography (CT) may show a "halo" sign — i.e., a zone of lower attenuation surrounding a pulmonary mass. CT scans may also show pulmonary cavitation, or "air crescent," formation [von Eiff et al., 1995]. Bronchoalveolar lavage (BAL) and sputum cultures vary in their diagnostic value, with reported positive results in 52% and 75%, respectively, in patients with proven pulmonary disease [Kaiser et al., 1998].

Studies have explored the value of using enzyme-linked immunosorbent assay (ELISA) to detect Aspergillus carbohydrate antigens in serum [Patterson et al., 1995] and an Aspergillus mitochondrial gene polymerase chain reaction (PCR)-ELISA assay on BAL fluids in diagnosing invasive aspergillosis [Jones et al., 1998]. In a study by Patterson et al. [1995], 54 patients had serum antigen levels >/=50 ng/ml as detected by ELISA; 19 of these patients had histologically proven disease and 16 had probable invasive aspergillosis. In a study by Jones et al., 1998, the Aspergillus mitochondrial gene PCR-ELISA was 100% sensitive and 100% specific for aspergillosis in neutropenic patients; all 12 neutropenic patients with definite or probable invasive aspergillosis had PCR-positive BAL fluids. PCR and ELISA have been used to detect serum galactomannan antigen in patients with invasive aspergillosis. Bretagne et al. [1998] found 19 of 20 PCR-positive serum samples to be positive for galactomannan. The results of these studies showed these methods to be highly predictive of infection, yielding an earlier diagnosis by noninvasive means.

Laboratory Diagnosis

The approach to laboratory diagnosis of fungal infections is similar to that for bacterial infections. Standard techniques for determining the type of fungal infection should include [Richardson and Warnock, 1997]:

• direct microscopic examination of clinical specimens to detect the presence of fungal components, including Gram stain, potassium hydroxide (KOH) preparation, India Ink stain, and others
• culture of clinical specimens to isolate and identify the fungus
• serologic tests for the detection of fungal antigens or antibodies in body fluids.

The specific procedures applied to the diagnosis of individual mycoses depend on the type of the infection and the status of the patient.

Direct Microscopic Examination

Direct microscopic examination of clinical specimens is an important first step in the laboratory diagnosis of fungal infections. Fungi are large enough to be seen in skin scrapings, tissue biopsy material, sputum, pus, and body fluids. The morphologic characteristics of the fungus are often distinctive enough to establish a diagnosis or narrow the possibilities [Howard et al., 1994; Joklik et al., 1992].

Samples collected for microscopic examination may be viewed directly or treated with special stains or reagents to improve the visibility of fungal elements. Below is a description of commonly used techniques [Richardson and Warnock, 1997].

Gram stain

This is the most widely used procedure in diagnostic microbiology. Bacteria are differentiated as Gram-positive (purple) or Gram-negative (red) based on their reaction to the staining procedure. Some fungi — mainly yeasts — stain Gram-positive [Brock and Madigan, 1991; Kern and Blevins, 1997]. The figure below shows a Gram stain demonstrating budding yeast cells and pseudohyphae characteristic of Candida albicans.

Gram Stain of a Clinical Specimen of Candida albicans

Courtesy of University of Washington; with permission.
Click on image for larger version.

Often, the presence of fungi in a clinical specimen is first noted following a routine Gram stain performed for a suspected bacterial infection. Yeast cells may be differentiated from bacterial cells in the specimen by their different forms and much larger size [Kern and Blevins, 1997].

Potassium hydroxide preparation

KOH preparation is used to examine specimens from skin and mucous membrane infections for the presence of fungi. The sample is mixed with a solution of 10% KOH. The alkaline KOH solution digests tissue debris but not the cell walls of fungi, thus allowing a better view of the fungal forms [Howard et al., 1994]. The figure below shows a KOH preparation demonstrating hyphae and yeast cells of C. albicans in a clinical sample obtained from a patient with oropharyngeal candidiasis.

KOH Preparation from a Case of Oropharyngeal Candidiasis

Courtesy of Michael R. McGinnis, PhD; with permission.
Click on image for larger version.

The addition of chemical brighteners, such as calcofluor white, to the KOH solution can further enhance the visibility of fungal structures. This compound binds to the chitin in fungal cell walls and fluoresces when exposed to ultraviolet light [Howard et al., 1994; Kern and Blevins, 1997].

India ink preparation

This method is used to detect encapsulated cells such as those of the yeast Cryptococcus neoformans in spinal fluid, urine, and other body fluids. India ink added to the specimen does not stain the polysaccharide capsule but provides a dark background against which encapsulated cells can be seen (see figure below) [Howard et al., 1994].

India Ink Preparation Demonstrating Encapsulated Yeast Cells Suggestive of Cryptococcus neoformans

Courtesy of Subhash K. Mohan, GAMS, MLT(CMLTD), ART(CSMLS); with permission.
Click on image for larger version.

A positive India ink prep is suggestive of, but not diagnostic of, C. neoformans because other yeasts, blood cells, and some artifacts may appear similar [Larone, 1995].

Other stains

Definitive diagnosis of most fungal infections requires evidence of tissue invasion as demonstrated by histopathology. The periodic acid-Shiff (PAS), Gomori methenamine-silver (GMS), hematoxylin and eosin (H&E), and Gridley stains are frequently used to prepare tissue samples for histologic examination and stain almost all fungi. With the PAS stain, fungi turn a red-violet fuchsia color and are easily discernible from lightly stained tissue cells in the background (see figure below) [Howard et al., 1994].

PAS Stain of Lung Tissue Demonstrating Invasion by Blastomyces dermatitidis

Courtesy of David Schlossberg, MD, FACP; with permission.
Click on image for larger version.

The GMS stain is the best stain for detecting fungi in tissue sections because it provides better contrast for screening and it also stains old and nonviable fungal elements better than the other two stains. Fungal cell walls are stained brownish-black, whereas the internal details of hyphae are charcoal gray. Most host tissue stains light green, although some tissue structures may pick up brownish-black or gray coloring (see following figure) [Collier et al., 1998; Larone, 1995].

GMS Stain Demonstrating Invasive Aspergillosis

Courtesy of Chandler and Watts [1987]; with permission.
Click on image for larger version.

Culture of Clinical Specimens

Microscopic examination of clinical specimens may quickly provide clinicians with enough information to support a presumptive diagnosis of fungal disease and to assist in the selection of treatment. However, culture of specimens is usually required to determine the identity of a fungal pathogen and make a definitive diagnosis [Howard et al., 1994].

Culture techniques

Culture procedures involve techniques for growing colonies of microorganisms in the laboratory. Fungi can be grown by methods similar to those used for bacteria. A portion of the specimen is placed on a culture medium and then incubated under environmental conditions that favor fungal growth [Kern and Blevins, 1997].

Fungi may grow on the several different types of media routinely used for culture in the microbiology laboratory. However, the best results are obtained with media designed specifically for fungi. Antibiotics are often added to the media to inhibit growth of bacteria and nonpathogenic fungal contaminants [Brooks et al., 1998; Kern and Blevins, 1997].

Several different types of culture media are available for different purposes. Commonly used media for primary recovery of fungi from clinical specimens include Saboraud dextrose agar, brain-heart infusion agar, and inhibitory mold agar. These media support growth of a wide range of fungi. Specialized media have been developed to selectively isolate specific organisms — e.g., birdseed agar assists in quick identification of C. neoformans. Other fungi may grow on this medium, but only C. neoformans produces a characteristic brown pigment. In some cases, fungi grown on a primary culture are subcultured on specialized media to enhance specific characteristics, such as spore formation or pigmentation of colonies. These features can assist in identification of the organism [Brooks et al., 1998; Howard et al., 1994; Kern and Blevins, 1997].

Growth rates for fungal cultures vary but generally are slow compared with bacterial cultures. Whereas some fungal colonies mature within 3-4 days, others may require 3-4 weeks. In general, yeasts grow faster than molds. The prolonged incubation time is a major limitation to the use of fungal cultures as a diagnostic tool. The results may become available too late to either help with the diagnosis or the choice of treatment [Howard et al., 1994; Kern and Blevins, 1997; Richardson and Warnock, 1997].

Identification of fungi grown in culture

Identification of fungi grown in culture begins with observation of their colonial morphology. Fungi can usually be characterized as yeasts or molds on the basis of texture. Yeast colonies are typically smooth because they produce no aerial hyphae. However, filamentous mold colonies have a fuzzy appearance. Additional features such as the size, topography, and color of the colony are helpful in narrowing the identity of the fungus to one of several groups [Howard et al., 1994; Kern and Blevins, 1997]. The approach to final identification of a fungus depends on whether the organism is a yeast or a mold [Kern and Blevins, 1997].

Species identification of molds is usually based on microscopic examination of their asexual conidia or sexual spores. A sample is obtained by removing a small portion of the colony and gently teasing it apart. Alternatively, the sticky side of clear tape is pressed onto the surface of the colony and gently lifted upward to remove the aerial hyphae. The sample is then placed in a drop of a blue dye, called lactophenol cotton blue (LPCB), which has been placed on a glass slide. A coverslip is applied, and the slide is examined [Sugar and Layman, 1997]. The following figure shows an LPCB preparation demonstrating the various morphological structures of Aspergillus fumigatus.

LPCB Preparation of Aspergillus fumigatus

Courtesy of Subhash K. Mohan, GAMS, MLT(CMLTD), ART(CSMLS); with permission.
Click on image for larger version.

Identification of yeasts is based on biochemical tests. These tests differentiate among yeasts based on their characteristic physiological reactions to various nutritional and environmental conditions and are similar to those used for bacteria. Several commercial test kits for fungi are available [Howard et al., 1994].

A rapid screening technique for C. albicans is the germ tube test. In this procedure, yeasts are placed in a small amount of serum and incubated at 37°C for 3 hours. Within this time period, most isolates of C. albicans produce tubelike projections, called germ tubes, which are the beginning of the formation of hyphae (see figure below). Although a few other species of Candida also produce germ tubes, most take longer than 3 hours [Howard et al., 1994; Kern and Blevins, 1997].

Candida albicans Positive Germ Tube Test

Courtesy of University of Washington; with permission.
Click on image for larger version.

Serologic Tests

Serology is the branch of laboratory medicine that studies blood and other body fluids for evidence of infection by measuring antigen and antibody levels. Essentially the same techniques are used in the diagnosis of bacterial and viral infections. Most serologic tests can be modified to detect either antibodies or antigens in a specimen. Several commercial test kits are available for the diagnosis of selected mycoses [Howard et al., 1994; Richardson and Warnock, 1997].

Detection of circulating antibodies

Serologic tests for the detection of fungal antibodies in the circulation are reserved for the diagnosis of subcutaneous and systemic mycoses. They may also be used to determine a prognosis and assess the response to treatment. Commonly used techniques include complement fixation, immunodiffusion, latex agglutination, and enzyme-linked immunosorbent assay [Howard et al., 1994; Richardson and Warnock, 1997].

Detection of antibodies is of limited value, especially in the acutely ill patient. Some patients may have antibodies in the absence of infection because they have been previously exposed to the fungus. This problem can be seen with Candida, which is part of the normal microbial flora. In most cases, a diagnosis cannot be based on analysis of a single specimen, unless the antibody titer is very high. Sequential tests to detect rising levels of antibodies in serum samples drawn 2 to 3 weeks apart can provide more meaningful data. However, this time requirement presents an obvious limitation. Thus, appropriate stains and cultures are usually more reliable [Howard et al., 1994; Richardson and Warnock, 1997].

Tests for the detection of antifungal antibodies are least useful in immunocompromised patients, in whom antibody production is impaired. In such patients, serologic tests for the detection of fungal antigen may be more useful [Howard et al., 1994; Richardson and Warnock, 1997].

Detection of fungal antigens

Detection of cryptococcal antigen via latex agglutination is an established procedure for the diagnosis of cryptococcosis, and several commercial test kits are available. Similar tests have been introduced for aspergillosis, candidiasis, and histoplasmosis. However, the tests for aspergillosis and candidiasis are seldom reliable. Clinical and other laboratory findings are more important in the diagnoses of most opportunistic mycoses. The table below describes the most widely used serologic tests for the diagnosis of mycoses [Howard et al., 1994; Richardson and Warnock, 1997].

Selected Serologic Tests Used in Mycology

Test	Description
Latex agglutination	A specific antibody coated onto latex particles will react with target antigen, resulting in visible clumping. Results can be read in 10-15 minutes. Kits are available to test for cryptococcal antigen. A variation using specific antigen can be used to detect antibodies in serum.
Immunodiffusion	Antigen and antibodies are allowed to diffuse toward each other through a clear agar gel in which they have been placed in separate wells. In a positive reaction, the formation of antigen-antibody complexes produces a visible precipitate.
Counter-immunoelectrophoresis	Immunodiffusion carried out in an electric field, which improves both speed and sensitivity of the reaction.
Complement fixation	Serum to be tested for antibody is mixed with a specific antigen. In the presence of antibodies, complexes will form and consume complement (substances involved in immune reactions). The amount of complement consumed is measured to determine the concentration of antibody.
Immunofluorescence staining	Specific antibodies tagged with a fluorescent dye are used to detect antigens in a sample.
Enzyme-linked immunosorbent assay (ELISA)	The binding of antibody to antigen is measured by binding of a second reagent, usually an antibody labeled with an enzyme that can be detected by a color reaction.
Radioimmunoassay	The technique is similar to ELISA, except that the second reagent is labeled with a radioisotope.

Data from Mims et al. [1998], Reese and Betts [1996], and Stedman [1989].

New Approaches

Although DNA probes, polymerase chain reaction, and other molecular biologic techniques are potentially valuable in detecting fungi, they are currently in developmental stages and are not generally available [Mims et al., 1998].

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