In this article we will discuss about the Classification and Laboratory Diagnosis of Fungal Infections.
Classification of Fungal Infections:
A. Superficial Mycoses:
Dermatophytosis caused by: Trichophyton, Epidermophyton and Microsporum species.
Pityriasis versicolor: Malassezia furfur.
White piedra: Trichosporon beigelii.
Black piedra: Piedraia hortaie.
Tinea nigra: Exophiala werneckii.
Candidiasis: Candida albicans and other species of Candida.
The specimens from this group include — skin, hair and nails.
B. Subcutaneous Mycoses:
Eumycetoma caused by: Madurella mycetomatis, Madurella grisea.
Black or dark grains: Leptosphaeria senegalensis, Exophiala jeanselmei, Pseudallescheria boydii.
White or yellow grains: Acremonium species.
Eumycetomas require to be differentiated from actinomycotic mycetomas.
1. Chromoblastomycosis: Fonsecaea pedrosoi (Pigmented fungi).
2. Sporotrichosis: Sporothrix schenckii.
3. Rhinosporidiosis: Rhinosporidium seeberi.
Specimens from this group include pus from abscesses, exudate from lesions, aspirated fluid from sinus tracts and tissue from biopsy.
C. Systemic Mycoses caused by ‘Pathogenic Fungi’:
1. Histoplasmosis: Histoplasma capsulatum.
2. Blastomycosis: Blastomyces dermatitidis.
3. Paracoccidioidomycosis: Paracoccidioides brasi- liensis.
4. Coccidioidomycosis: Coccidioides immitis.
D. Systemic Mycoses caused by ‘Opportunistic Fungi’:
1. Cryptococcosis: Cryptococcus neoformans.
2. Aspergillosis: Aspergillus fumigatus and other aspergillus species.
3. Zygomycosis: Mucor, Rhizopus and Basidiobolus species.
4. Systemic candidiasis: Candida albicans.
Specimen from this group include pus or exudate, cerebrospinal fluid, respiratory secretions, bone marrow, blood, urine and tissue from biopsy or autopsy.
Laboratory Diagnosis of Fungal Infections:
I. Specimens:
1. Skin scrapings, nail clippings and hairs can be transported in an envelope, Petri dish, or other convenient conveyance.
2. Specimens from mucous membrane may be directly inoculated in culture medium or smeared on clean slide by swab or culture loop.
3. Scrapings, crusts, aspirated pus and tissue biopsies are to be collected aseptically from subcutaneous infections.
4. In suspected systemic infections, specimens are to be collected from as many sites as possible, such as blood and CSF.
Specimen processing:
The specimen is divided into two parts, one for microscopy and other for culture. Yeasts are handled in the same way as bacteria. Moulds, however, require some special procedures.
II. Examination:
Microscopy:
1. Potassium hydroxide (KOH) mount:
KOH dissolves keratin and cellular material but does not affect fungi. Specimens are placed on a slide to which a drop of 10-20% potassium hydroxide is added and then covered by a cover slip and left for 20 minutes in incubator at 37°C to digest keratin. Microscopical examination shows fungal structure.
2. Staining technique:
Gram stain, Papanicolaou (usually performed in cytopathology laboratory), Periodic acid-Schiff (PAS) staining and methenamine silver staining stain most fungi well. Giemsa staining is useful for yeasts and cells of Histoplasma capsulatum because of their small size. All fungi are gram-positive though of little value.
3. Direct immunofluorescence:
DFAT is useful to identify fungi in tissues and smears.
4. Histology:
Examination of biopsy specimens of tissue provide firm evidence of invasive disease. Tissue sections prepared from paraffin block are stained by Gomori methenamine silver and periodic acid-Schiff stain. Such staining indicates only the presence of fungi in tissues and not the species.
Antigen detection:
Detection of cryptococcal polysaccharide antigen in CSF and serum by latex agglutination test is routinely done. The test is sensitive, specific and simple to perform. Methods for detection of Candida and Histoplasma antigens have also been described.
Culture and isolation:
1. Culture media:
Pathogenic fungi grow on Sabouraud dextrose agar (pH 5.6, slight acidic pH which does not allow bacterial growth) contains 4% dextrose, 1% peptone, and 2% agar. Chloramphenicol (50 mg/L), cycloheximide (500 mg/L), or other antibiotic is often added to the medium to further prevent bacterial or saprophytic fungal contamination.
2. Technique:
Two cultures with the specimens are done and incubated at room temperature (25°C) and at body temperature (37°C) to reveal dimorphism. Tube, Petri dish and slide are used for culture. Slide culture is useful for studying morphology of fungus. Cultures are incubated for at least 2 to 3 weeks and in some cases up to 6 weeks.
3. Identification:
Culture provides an unequivocal evidence of fungal infection.
(a) Macroscopic appearance (colony character) (Fig. 14.1):
(i) Dimorphic fungi produce colonies of 1-2 mm diam, delicate hyphae which give the mould form of the colonies a cobweb or hair-like appearance (Fig. 14.1A).
(ii) Trichophyton rubrum produces wine-red diffusible pigment.
(b) Microscopic morphology (Tease mount):
A small portion of the colony including some of the surface agar is dug out with a pair of dissecting needles or pointed applicator sticks. The colony fragment is placed on a microscope slide in a drop of lacto-phenol aniline blue.
The colony is teased apart using dissecting needles, and then a cover slip is positioned over the drop and gently pressed on the surface with the eraser end of a pencil to facilitate microscopic examination.
Findings:
Yeasts are unicellular. On the other hand, moulds are filamentous (hyphae), produce specialised conidia, and grow slowly.
The following fungal structures may be seen:
(i) Mycelium: Presence of septate or aseptate hyphae (Fig. 14.2), or pseudo hyphae.
(ii) Mycelium may be vegetative (portion extending into culture medium) or aerial (portion above the medium/substrate).
(iii) Presence of specialised structures, e.g. chlamydospores, or yeasts, etc. may be present.
(c) Nucleic probes:
Genetic probes are available for detection of Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum.
Indirect methods based on host immune response:
(a) Skin testing:
Skin test once a popular diagnostic test is now almost abandoned because of false-positive results. However, those tests are useful to study patient’s immune status.
(b) Serologic tests:
Serologic tests like ELISA for histoplasmosis, latex agglutination test and CFT have been devised.