Laboratory Procedures for the Diagnosis of Fungal Infection Flashcards
Laboratory Procedures for the Diagnosis
of Fungal Infection
•combination of clinical observation and laboratory investigation
•Superficial and subcutaneous fungal infections often produce characteristic lesions
•For systemic mycosis –definitive diagnosis is almost entirely based on results of laboratory investigation
Laboratory Procedures
•Quality specimen for mycotic laboratory investigation
•collection of adequate clinical specimens for investigation
•Good storage
•Quality information needed for suspicion of mycotic investigation
•Signs and symptoms
•Underlying illness
•recent travel
•previous residence abroad
Mycotic diagnosis
•Three main broad approaches
•the microscopic detection of the etiologic agent in clinical material
•its isolation and identification in culture
•the detection of either a serologic response to the pathogen or some marker of its presence, such as a fungal cell constituent or metabolic product
•New diagnostic procedures based on the detection of fungal
DNA in clinical material are presently being developed
Direct Microscopic Examination of Clinical
Specimens
•Microscopic examination of skin scrapings or other superficial material can reveal a fungal organism in a matter of minutes
•Guide treatment decisions
•Guide whether an organism recovered later in culture is a contaminant or a pathogen
•Selecting the most appropriate culture conditions to recover organisms visualized on direct smear
•Generally less sensitive
•Culture should be performed
Keratinised dermatologic specimens, sputum and other lower respiratory tract specimens, and minced tissue samples can be examined after treatment with 10–20% Potassium hydroxide (KOH)
•wet preparation
•without stain or
•With stain => lactophenol cotton blue, methylene blue (reagents) with low power light microscope
•With stain => calcofluor white (chemical brightener) stains fungal cell wall with fluorescent microscope
•India ink (negative staining)- reveals encapsulated Cryptococcus neoformans cells in CSF
•Gram stain
•Giemsa stain and Wright’s stain can be used to detect Histoplasma capsulatum in bone marrow preparations or blood smears
•Papanicolaou stain
Draw backs of direct microscopy
•Quality of the specimen
•Age of the specimen
•the extent of the disease process
•the nature of the tissue being examined
•experience of the microscopist
Draw backs of direct microscopy
•Quality of the specimen
•Age of the specimen
•the extent of the disease process
•the nature of the tissue being examined
•experience of the microscopist
Microscopic Examination of Clinical
Specimens
•Histopathologic examination
•One of most reliable methods of establishing the diagnosis of subcutaneous and systemic fungal infections
•Depends on:
•abundance
•distinctiveness of its appearance
•Hematoxylin and eosin
•Methenamine-silver (Grocott or Gomori)
•Periodic acid-Schiff (PAS)
Draw back of Microscopic Examination of Clinical Specimens
•seldom permits the precise fungal genus involved
to be identified
•Immunohistochemical methods have more specificity with use of Monoclonal antibodies
•Useful for Aspergillus species or members of the order
Mucorales at present
Culture
•Isolation in culture will permit most pathogenic fungi to be identified
•Most pathogenic fungi are not fastidious hence can grow on any media
•Although growth can be slow to appreciate finite fungal structures
•Culture media:
•Sabouraud’s dextrose agar,
•Potato dextrose agar,
•Potato flakes
•Brain heart infusion specially for fastdious Histoplasma capsulatum
•Additives such as chloramphenicol , gentamicin and cycloheximide to suppress bacteria
A variety of chromogenic agars that incorporate multiple chemical dyes in a solid medium can be used giving different colors based on substrate utilisation and differential update of these chromogenic compounds
•CHROMagar Candida
Temperature for Culture
•The optimum growth temperature for most pathogenic
fungi is around 30°C
•Material from patients with a suspected superficial infection should be incubated at 25–30°C, because of dermatophytes
•Material from subcutaneous or deep sites should be incubated at two temperatures, 25–30°C and 37°C
•H. capsulatum, B. dermatitidis and Sporothrix schenckii, are dimorphic and the change in their growth form, depending on the incubation conditions, is useful in identificati
Time for culture
Maximum time of about 4 week should be allowed for some slow growing fungi but fast growers such as Candida and Aspergillus will grow in 1-3 days
•Some fungi are obligate pathogens: H. capsulatum or Trichophyton rubrum
•While some are opportunistic: A. fumigatus or C. albicans
No isolate should be dismissed as a contaminant without careful consideration of the clinical condition of the patient, the site of isolation, the method of specimen collection, and the likelihood of contamination !!!!!!
•Draw backs
•the failure to recover an organism does not rule out
the diagnosis
•Causes?????
Blood culture
•Candida species are more readily recovered from blood than are dimorphic fungi and moulds
•lysis-centrifugation method for C. neoformans and H. capsulatum
•Semi-automated agitation blood culture systems
Fungal Identification
•Phenotypic identification
•macroscopic and microscopic morphology
•Macroscopic characteristics, such as colonial form, surface color and pigmentation
•Microscopy: on the form and arrangement of the conidia and the structures on which they are produced
•shape of the cells, the presence of a capsule around the cells, the production of hyphae or pseudohyphae, and the production of chlamydospores
•Spores can be induced for identification
•Slide culture????
•Biochemical test
•assimilation and fermentation of sugars
•the assimilation of nitrate and urea
•Commercial identification systems, such as the API 20C, API 32C, Vitek Yeast Biochemical Card
•Simple rapid tests
•germ tube test for C. albicans, which can be performed in less than 3 hrs
•the urease test for Cryptococcus neoformans
•rapid trehalose test for C. glabrata
Serologic testing
•Serologic testing often provides the most rapid means of diagnosing a fungal infection
•Assists in definitive diagnosis
•Assess status of previously diagnosed mycosis
•Determine efficacy of therapy
•Detection of
•antibodies to specific fungal pathogens
•fungal antigens
•Serologic testing must be interpreted alongside other clinical and laboratory investigation
Antibodies testing in diagnosing endemic fungal infections, such as histoplasmosis and coccidioidomycosis in immunocompetent persons
•More useful with paired serum specimens (acute and convalescent) are obtained
•Immunodiffusion =>Ab
•Complement fixation =>Ab
•Latex agglutination =>Ab &Ag
•Enzyme-linked immunosorbent assay (ELISA) =>Ab &Ag
•Antigen detection
•cryptococcosis and histoplasmosis
•Aspergillosis - Low concentrations of galactomannan, a major cell wall component
•Urine, serum, and bronchial lavage fluid
•A challenge is lack of sensitivity/ specificity/cross-reactions
•Candida antigens
•mannan (a heat-stable cell wall component), enolase, proteinase, and other immunodominant antigens
•Latex agglutination for cryptococcosis; serum and CSF Antigens
•(1–3)-Beta-D-glucan is a cell wall component of many fungi, including Aspergillus and Candida spp.
•NOT in Cryptococcus species or zygomycetes
