Practical Medical Mycology Guide PDF

Practical part of Medical Mycology Method of Fungi isolation from clinical specimens:  Fungal cultures are microbiology laboratory tests to detect the presence of fungi in specimens taken from patients.  The laboratory employs optimal conditions to grow and identify any fungus present in the spec...

Practical part of Medical Mycology Method of Fungi isolation from clinical specimens:  Fungal cultures are microbiology laboratory tests to detect the presence of fungi in specimens taken from patients.  The laboratory employs optimal conditions to grow and identify any fungus present in the specimen.  The specimen is cultured on various agar media and then incubated and examined.  Specimen could be the skin scrapping, nail scrapping, infected hair etc.  Plate exposure method:  Useful method for airborne fungi.  Certain molds are likely to get their spores into the air and these spores may be serve as an infective agent of some allergies.  These can be isolated by —> plate containing PDA is exposed to air for few minutes — -> covered ànd incubate at 25oC or room temperature for few days —-> observe colonies. Identification of fungi: Lactophenol Cotton Blue (LPCB) Staining - Lactophenol Cotton Blue (LPCB) Staining is a simple histological staining method used for the microscopic examination and identification of the fungal spore cell wall which is made up of chitin. - The stain will give the fungi a blue-colored appearance of the fungal spores and structures, such as hyphae. 1  KOH Preparation Test - Potassium hydroxide (KOH) preparation is used for the rapid detection of fungal elements in clinical specimen, as it clears the specimen making fungal elements more visible during direct microscopic examination. - KOH is a strong alkali. When specimen such as skin, hair, nails or sputum is mixed with 20% w/v KOH, it softens, digests and clears the tissues (e.g., keratin present in skins) surrounding the fungi so that the hyphae and conidia (spores) of fungi can be seen under microscope. 1- Candida Laboratory Diagnosis of Candidiasis Specimen and specimen preparation  Oral swabs and scrapings from lesions, vaginal swabs, blood, spinal fluid, tissue biopsies, urine. Microscopic Examination  Examine the specimens by Gram staining for identification of pseudohyphae and budding cells  Skin or nail scrapings can be analyzed using KOH wet mount for observation of the pseudohyphae and formation of a germ tube. Cultural examination  The specimens can be examined by culture methods using fungal media and/or bacterial media at room temperature or at 37°C.  The colonies are examined for the formation of pseudohyphae.  Candidemia is primarily diagnosed through blood cultures. 2  2- Cryptococcus spp Morphology of Cryptococcus neoformans  Cryptococcus neoformans is a yeast fungus, producing yeast cells during reproduction.  The yeast cells are dry, mildly encapsulated, and light, making them easy to aerosolize. Cultural characteristics of Cryptococcus neoformans  In culture, they produce whitish mucoid colonies within 2-3 days.  They have spherically budded yeast cells of 5–10 μm in diameter with a thick non staining capsule surrounding it.  They lack hyphae.  It has the ability to grow at 37°C, producing laccase, which is a phenol oxidase that catalyzes the formation of melanin from phenolic substrates such as catecholamine, which can be detected by biochemical characterization. Laboratory Diagnosis of Cryptococcus neoformans Staining and Microscopic examination: Gram staining  The cells of Cryptococcus neoformans may appear round Negative staining  Using India Ink, that particles of ink pigments cannot enter the cell capsule surrounding the yeast cells. This forms a zone of clearance known as a halo, around the cells. This is a quick method for the identification of Cryptococcus neoformans. 3 Cultural examination  Culture examination uses Sabouraud Dextrose Agar (SDA).  The yeast develops colonies within a few days on most cultural media at room temperature or 37°C.  Detection of urease production in the culture media.  Laccase of C. neoformans produces melanin in the cell walls, and colonies develop a brown pigmentation. Serological Examination Antigen detection  Detection of the capsular polysaccharide antigen of Cryptococcus is the idlest diagnostic test by using spinal fluid or serum or urine by the application of enzyme immunoassays or agglutination of latex particles coated with antibodies to the polysaccharide antigen.  This test is sensitive and specific for antigen detection in cases of cryptococcal meningitis. Antibody Quantification  Antibodies produced against the polysaccharide capsule antigen can be quantified. 3- Aspergillus niger Laboratory Diagnosis of Aspergillus niger  Microscopic examination to reveal the dark brown rough-edged conidia spores. 4  Cultural examination using Potato dextrose Agar, Czapek yeast Agar and Malt Yeast Agar.  Genomic sequencing of the fungi can be done for identification and differentiation from other fungi. Cultural Characteristics of Aspergillus niger  Generally, they have a cottony appearance; initially white to yellow and then turning black. The reverse is white to yellow.  Malt Extract Agar – an incubation for 7 days at 25ºC and 37ºC producing slightly brown colonies smooth-walled colonies of conidia.  Czapek Yeast Agar – after 5 days of incubation at 25ºC and 37ºC, they produce black colonies with wooly smooth-walled colonies of conidia. 4- Blastomyces dermatitidis Diagnosis  Fungal cultures and smear  Blastomyces urine antigen 5   If blastomycosis is suspected, a chest x-ray should be taken.  Molecular diagnostic tests (eg, polymerase chain reaction [PCR]) are becoming available. 5- Dermatophytes Diagnosis and identification  Usually, dermatophyte infections can be diagnosed by their appearance.  However, a confirmatory rapid test can also be performed, by using lesion sample from the nail, skin, or scalp and transferring it to a slide. Potassium hydroxide (KOH) is added to the slide and the sample is examined with a microscope to determine presence of hyphae.  In the culture, characteristic septate hyphae can be seen  A special agar called Dermatophyte Test Medium (DTM) can be used to grow and identify dermatophytes.  It is incubated at room temperature for 10 to 14 days. If the fungus is a dermatophyte, the medium will turn bright red. If the fungus is not a dermatophyte, no color change will be noted. 6