Laboratory Diagnosis

Specimen Collection and Transport

See General Considerations for the Laboratory Diagnosis of Fungal Infections.

Specimen Processing

See General Considerations for the Laboratory Diagnosis of Fungal Infections.

Direct Detection Methods

Stains. Specimens submitted for direct microscopic examination that contain organisms in this group demonstrate septate hyphae that usually shows evidence of dichotomous branching, often of 45-degrees (see Figure 50-14). In addition, some hyphae may show the presence of rounded, thick-walled cells. Although often considered to represent an Aspergillus species, these cannot be reliably distinguished from hyphae of Fusarium spp., Pseudallescheria boydii, or other hyaline molds.

Antigen-Protein. Antigen-protein-based assays are beginning to be used to monitor patients who are at high risk for developing invasive fungal infections. One of these assays, the galactomannan assay, specifically targets antigens of Aspergillus species, because this is the most common cause of invasive fungal infections caused by the hyaline septate molds (i.e., hyalohypbomy-cosis).107,153 Conversely, the beta-glucan assay is designed to detect antigens common to all clinically important fungi.97,99153 How these tests will be used in the future and how they will compare with nucleic acid amplification tests remain to be determined.

Nucleic Acid Amplification. Nucleic acid amplification assays are not commonly performed for the detection or identification of these fungi. However, a variety of both broad-range (i.e., those that detect all fungi) and species-specific assays have been developed and in specialized centers may be used for patient care.

Cultivation. Because aspergilli are recovered fre-quendy, it is imperative that the organism be demonstrated in the direct microscopic examination of fresh clinical specimens and/or that it be recovered repeatedly from patients having a compatible clinical picture to ensure that the organism is clinically significant. Cor relation with biopsy results is the best way of establishing the significance of an isolate. Most species of Aspergillus are susceptible to cycloheximide. Therefore, specimens submitted for its recovery or subcultures should be inoculated onto media that lacks this ingredient

Aspergillus fumigatus is the most commonly recovered species from immunocompromised patients; moreover, it is the spedes most often seen in the clinical laboratory. In addition, Aspergillus flavus is sometimes recovered from immunocompromised patients and represents a frequent isolate in the clinical microbiology laboratory. The recovery of A. fumigatus or A.flavus from surveillance (nasal) cultures has been correlated with subsequent invasive aspergillosis.141 The absence of a positive nasal culture does not preclude infection, however. Aspergillus niger is seen commonly in the clinical laboratory, but its association with clinical disease is somewhat limited; this organism is a cause of fungus ball and otitis externa. Aspergillus terreus is a significant cause of infection in immunocompromised patients, but its frequency of recovery is much lower than the previously mentioned species. It is, however, important to correcdy identify this species, because it is innately resistant to ampicillin B.

Approach to Identification

Aspergillus. Aspergillus fumigatus is a rapidly growing mold (2 to 6 days) that produces a fluffy to granular, white to blue-green colony. Mature sporulating colonies most often exhibit the blue-green powdery appearance. Microscopically, A. fumigatus is characterized by the presence of septate hyphae and short or long conidiophores having a characteristic "foot cell" at their base. The tip of the conidiophore expands into a large, dome-shaped vesicle that has bottle-shaped phialides covering the upper half or two thirds of its surface. Long chains of small (2 to 3 pm in diameter), spherical rough-walled, green conidia form a columnar mass on the vesicle (Figure 50-58). Cultures of A. fumigatus are thermo-tolerant and are able to withstand temperatures up to 45° C.

Aspergillus flam is a somewhat more rapidly growing species (1 to 5 days) and produces a yellow-green colony. Microscopically, vesicles are globose and phialides are produced direcdy from the veside surface (uniserate) or from a primary row of cells termed metulae (biserate). The phialides give rise to short chains of yellow-orange elliptical or spherical conidia that become roughened on the surface with age (Figure 50-59). The conidiophore olA.flavusisdiso rough.

Aspergillus niger produces darkly pigmented roughened spores, but the hyaline septate hyphae like other aspergilli (i.e., it is not dematiaceous). Aspergillus niger produces mature colonies within 2 to 6 days. Growth

Aspergillus 400x
Figure 50-58 Aspergillus fumigatus conidiophore and conidia (400x).
Vesiculas Esfericas

Figure 50-59 Aspergillus flavus showing spherical vesicles (A) that give rise to metulae (B) and phialides (C) that produce chains of conidia (750x).

Figure 50-59 Aspergillus flavus showing spherical vesicles (A) that give rise to metulae (B) and phialides (C) that produce chains of conidia (750x).

begins initially as a yellow colony that soon develops a black, dotted surface as conidia are produced. With age, the colony becomes jet black and powdery while the reverse remains buff or cream color; this occurs on any culture medium. Microscopically, A. niger exhibits septate hyphae, long conidiophores that support spherical vesicles that give rise to large metulae, and smaller phialides (biseriate), from which long chains of brown to black, rough-walled conidia are produced (Figure 50-60). The entire surface of the vesicle is involved in sporulation.

Aspergillus terreus is less commonly seen in the clinical laboratory; it produces colonies that are tan and resemble cinnamon. Vesicles are hemispherical, as seen microscopically, and phialides cover the entire surface and are produced from a primary row of metulae (biserate). Phialides produce globose to elliptical conidia arranged in chains. This species produces larger cells, aleurioconidia, which are found on submerged

Aleurioconidia

Figure 50-61 Aspergillus terreus showing typical head of aspergillus and aleurioconidia (arrow) found on submerged hyphae of this species (SOOx).

Aspergillus Terreus

Figure 50-60 Aspergillus niger showing larger spherical vesicle that gives rise to metulae, phialides, and conidia (750x).

hyphae (Figure 50-61). Refer to the work by Kennedy and Sigler67 for further information regarding these and other species.

Fusarium. Colonies of Fusarium grow rapidly* within 2 to 5 days, and are fluffy to cottony and may be pink, purple, yellow, green, or other colors, depending! on the species. Microscopically the hyphae are small and septate and give rise to phialides that produce either single-celled microconidia, usually borne in gelatinous heads similar to those seen in Acremonium (see Figure 50-39) or large multicelled macroconidia that are sickle* or boat-shaped and contain numerous septations (Figure 50-62). It is common to find numerous chlamydoconidia produced by some cultures of Fusarium. The most common medium used to in' duce sporulation is cornmeal agar. Keys for identify cation of species by Fusarium are based on on potato dextrose agar.

Figure 50-60 Aspergillus niger showing larger spherical vesicle that gives rise to metulae, phialides, and conidia (750x).

Figure 50-61 Aspergillus terreus showing typical head of aspergillus and aleurioconidia (arrow) found on submerged hyphae of this species (SOOx).

Molds With Thick Aleurioconidia

Figure 50-64 The mycelial form of Coccidioides immitis showing numerous thick-walled, rectangular or barrel-shaped (arrows) alternate arthroconidia (SOOx).

Figure 50-62 Pusarium spp. showing characteristic raulticelled, ijckie-shaped macroconidia (500x).

Figure 50-64 The mycelial form of Coccidioides immitis showing numerous thick-walled, rectangular or barrel-shaped (arrows) alternate arthroconidia (SOOx).

Geotrichum Candidum
Figure 50-63 Geotrichum candidum showing numerous arthroconidia. Note that arthroconidia do not alternate with a dear (dysjunctor) cell as in the case of C. immitis (430x).
Figure 50-65 Penicillium spp. showing typical brushlike conidiophores (penicillus) (430x).
  1. Geotrichum often initially appears as a white to cream yeastlike colony; some isolates may appear as white, powdery molds. Hyphae are septate and produce numerous rectangular to cylindrical to barrel-shaped arthroconidia (Figure 50-63). Arthro-condia do not alternate, but are contiguous as contrasted with Coccidioides immitis (Figure 50-64). Blastoconidia are not produced.
  2. Colonies of Ammonium are rapidly growing and also may appear yeastlike when initial growth is observed. Mature colonies become white to gray to rose or reddish orange in color. Microscopically, small septate hyphae that produce single, unbranched, tubelike phialides are observed. Phialides. give rise to clusters of elliptical, single-celled conidia contained in a gelatinous cluster at the tip of the phialide (see Figure 50-39).
  3. Colonies of Penicillium are most commonly shades of green or blue-green, but pink, white, or other colors may be seen. The surface of the colonies may be velvety to powdery because of the presence of conidia. Microscopically, hyphae are hyaline and septate and produce brushlike conidiophores (i.e., a penicillus). Conidiophores produce metulae from which phialides producing chains of conidia arise (Figure 50-65). Penicillium marneffei, a particularly virulent species in their genus, is discussed in the section on hyaline, septate, dimorphic molds.
  4. Colonies of Paecilomyces spp. are often velvety, tan to olive brown, and somewhat powdery. Colonies of P. lilacinus exhibit shades of lavender to pink. Microscopically, Paecilomyces resemble a Penicillium in that a penicillus is formed. However, the phialides of Paecilomyces are long.
Scopulariopsis Brumptii Images

Figure 50-66 Paedlomyces spp. showing long, tapering, delicate phialides (arrow).

Scopulariopsis Brumptii

Figure 50-67 Scopulariopsis spp. showing a large penicilllus (A) with echinulate conidia (B) (43Ox).

brevicaulis. Scopulariopsis brumptii is a dematiaceoug species and is occasionally recovered in the clinfcgj laboratory; it has been reported to cause brain abscesg in a liver transplant patient.104

Serodiagnosis

The use of Aspergillus serology is limited to assistance in the diagnosis of bronchopulmonary aspergillosis and fungus ball. Serology is currentiy not of value for the diagnosis of disseminated aspergillosis.

hyaline, septate, dimorphic molds:

systemic mycoses --

Genera and Species to Be Considered! Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, Paracoccidioides brasillensis, Pénicillium marneffei, and Sporothrix schenckii

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Bacterial Vaginosis Facts

Bacterial Vaginosis Facts

This fact sheet is designed to provide you with information on Bacterial Vaginosis. Bacterial vaginosis is an abnormal vaginal condition that is characterized by vaginal discharge and results from an overgrowth of atypical bacteria in the vagina.

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