The laboratory diagnosis of fungal infection begins with the appropriate sample collection and transportation.In most fungal infections, identifications are primarily based on the assessment of colony morphology and microscopic characteristics.Key biochemistry tests may be required to distinguish genes and species.In some cases, serological studies using lateral flow assay are needed to establish a differential diagnosis,cytospin method and molecular techniques such as Nucleic acid probe tests with increased frequency are also used to provide early confirmation in suspected cases of deep – seated mycoses.Non – cultural methods & automated system are also available for diagnosing fungal infections.
COLLECTION AND TRANSPORT OF SPECIMEN
For the laboratory diagnosis of fungal infections such as fungi nail infections,foot fungal infection,yeast infection prevention and fungal infection treatment,different samples can be received in the laboratory.Doctors, nurses, ward staff and laboratory technicians must work together to develop protocols that ensure proper and prompt collection of samples and the selection of appropriate collection devices,transport containers, specimen labeling and complete application forms are important considerations to ensure the correct diagnosis of fungal infections
Transport conditions for fungal infection diagnosis
|Specimen||Condition of Transport|
|Tissue biopsy||The sample should not be frozen or dehydrated before culture|
|Urine||If a delay of more than 2 hours is expected, refrigerate at 4 ° C.|
|Blood||Biphasic agar broth bottles specifically designed for fungal cultures|
|CSF||If the delay is anticipated, the sample must be left at room temperature.|
|Bronchoscopy Fluid||Sterile Screw capped container|
|Sputum||Sterile Screw capped container|
The specimen should be transported to the laboratory as soon as possible.In general, specimens that are not processed immediately are held at room temperature (for urine if delays exceed 2 hrs at the specimen should be refrigerated at a temperature of 4 ° C).Cryptococcus neoformans, Histoplasma capsulatum and Blastomyces dermatitidis do not survive in ice or frozen specimens.
HOW TO PROCESS FUNGAL SPECIMEN
For common fungal infections,specimens should be processed immediately after reception of the sample. The direct wet mount or swabs are prepared and the sample is inoculated on specific culture media for culture.
Direct Examination of Fungal specimen
Almost all samples are processed by direct macroscopic and microscopic examination. This provides the presumed diagnosis for the physician and also helps in the selection of appropriate culture media.
The following are various methods for direct examinations of fungal specimen
- Direct wet mount of specimen
- Lactophenol cotton blue (LPCB) mounts
- India Ink
- KOH/calcofluor mounts
- Frozen section of tissue biopsies
- Modified Kinyoun Acid Fast Stain for Nocardia
|Direct Microscopic Observations||Presumptive Identification|
|Hyphae with irregular size (6-50 μm),Ribbon Like and without septa||Zygomycetes (Phycomycetes) rhizopus-Mucor|
|Hyphae relatively small (3 – 6 μm) and regular in size, dichotomous branching at an angle of 45 ° with distinct cross septa||Aspergillus spp|
|Small hyphae (2 – 3 μm) and regular, sometimes seen branching with rectangular arthrospores found only in skin, nail scraping and hair||Microsporum spp|
|Regular hyphae with diameter (3-6 µm), parallel walls, irregular branching, septate, dark yellow, brown or hyaline.||Phaeohyphomycosis spp|
|Hyphae,distinct points of constriction simulating link sausages (pseudohyphae), with budding yeast forms (blastospores) often seen||Candida spp|
|Yeast forms, cell are spherical and irregular in size (5-20 µm) classically with a thick polysaccharide capsule (not all cells are encapsulated), with one or more buds attached by a narrow constriction||Cryptococcus spp|
|Small budding yeast, which are relatively uniform in size (3-5 µm) with a single bud attached by a narrow base, extracellular or within macrophages||Histoplasma capsulatum|
|Yeast forms with large cells (8-20 µm) appearing to have a thick, double contoured wall, with a single bud attached by a broad base||Blastomyces dermatitidis|
|Large and irregularly sized (10-50 µm) thick walled spherules, many of which contain small (2-4 µm) round endospores||Coccidioides immitis|
Wet Mounts Preparation
The microslide techniques and tease mount, transparency tape method are commonly used methods for fungal microscopic examination.
As for the mold colony,It is mounted in a drop of Lactophenol cotton blue stain on a glass slide and microscopically examined.
The specimen are directly mounted in 10% and 40% Potassium Hydroxide for nail and skin specimens respectively. Skin and nail samples are mounted on a glass slide to which two drops of Potassium hydroxide preparation is added and kept for sometime. KOH helps in dissolving the epithelial cells giving a clear fungal visibility.
This can be added to the specimens such as exudates or spinal fluids which provide a dark background that highlights hyalines and capsular material (halo effect).It should therefore be used to examine specimens suspected of containing Cryptococcus neoformans.White blood cells may differ from Cryptococcus neoformansdue to the irregular edge of the halo and pale cell wash.Some laboratory don’t routinely offer India ink preparation but instead offer the Cryptococcus Antigen Test which is serological test.
Fungi Gram stain
Gram staining is usually poor when used to stain fungal specimen.Gram staining method may be used when smears of Malassezia,Candida species and Sporothrix are examined but should not be used to detect yeast from other dimorphic fungi.Also Gram stain can be used to detect the filaments of Nocardia and Actinomyces, which causes clinical symptoms similar to mycotic infections.
Modified staging Stain for Nocardia
This method is used to identify Nocardia species
- Start by Making a thin smear of the specimen to be stained
- Fix it in methanol. You can use a positive control smear organism of Nocardia asteroides and a negative control smear of Streptomyces species.
- Flood the smear with Kinyoun carbolfuchsin for 5 minutes (Do not heat)
- Rinse the smear with water
- Decolorize the smear with 50% ethanol until excess carbolfuchsin is removed.
- Rinse the smear with water
- Decolorize with 0.5% (aqueous) Sulphuric acid for 3 minutes
- Rinse with water
- Flood the slide with 1% (aqueous) methylene blue for 1 minute.
- Rinse with water and examined microscopically
Selection and Inoculation of fungal culture media
Usually two types of culture media are used that is non – selective growth media such as brain heart infusion which allows the growth of all clinically relevant fungi.The use of sabouraud’s dextrose agaras the primary recovery medium is discouraged because it is insufficiently rich to recover certain fastidious pathogenic species, especially those dimorphic fungi.It is recommended to use growth media like Potato flake agar (PFA), inhibitory mold agar (IMA) or a combination of sabouraud dextrose agar with Brain heart infusion (SABHI) agar.
Sabouraud’s dextrose agar is sufficient for the recovery of dermatophytes from cutaneous samples or vaginal culture yeasts.Czepak ‘s agar can be used for subculture of aspergillus species if colony morphology is an important criteria for identifying any given unknown isolate.
For more fastidious dimorphic fungi such as Blastomyces dermatitidis and histoplasma capsulatum, an enrich agar like IMA or SABHI is used and in particular for Histoplasma capsulatum media with the addition of 5 – 10 percent sheep blood is recommended.
Aspergillus fumigatus and Cryptococcus neoformans can be partially or completely inhibited with antibiotic cycloheximide, so a non – selective media should always be used in parallel.
Incubation of fungal culture
Fungal sample are cultured each in two set of culture media and incubated at two different temperatures at 30 °C (Room temperature) and at 35°C
All fungal cultures are incubated for a minimum of 30 days before being discard as negative or no growth.The choice between the use of culture tubes or plate is optional. For tube, the media is poured in thick slants which prevent dehydration during prolonged incubation period. After the medium is inoculated, do not screw down the cap too tightly because fungi require breathing.
Culture media in tubes have the advantage of easy transport, while limitation is difficult to prepare stained mounts for microscopic examination and petri dishes have the advantages of providing a larger surface for growth that leads to better separation of the colony and makes cultures easier to examine and subculture.Also tease mounts or tape preparations for transparency are made from plate cultures.
The disadvantage with petri dishes is that the plates can be dehydrated during prolonged incubation period.In order to prevent the drying of the plates,They are placed in a sealed, moisturized polyester or the edges are sealed with oxygen permeable tape.
LABORATORY PROCEDURE FOR PRESUMPTIVE IDENTIFICATION OF FUNGI
After the culture plates have shown fungal growth of possible fungi, the colonies are identified by colony characteristic and morphology. A Lactophenol cotton blue mount is also prepared from growth culture and examined microscopically.
Colonies with a creamy,smooth,viscous or pasty look, could probably be yeast hence should be considered.Dematiaceous molds produce dark colonies.Gray to black mycelium growth and the colony reverse is black.For molds that grow within 3 – 5 days have a distinct border and on the surface are white or patel.One dimorphic species should be considered for molds growing in 7 – 14 days or having a cobweb aerial mycelium.
AUTOMATED SYSTEMS FOR FUNGAL IDENTIFICATION
The only commercially available current system is the applied Biosystems, the Microseq OZ large subunit r DNA sequencing kit. Micro scan also offers the identification kit for Candida species and candida albicans test such as threelac candida but not for other fungi isolates.
MOLECULAR TECHNIQUES FOR FUNGAL IDENTIFICATION
Nucleic acid probe assays are used with increasing frequency to confirm early culture, especially in deep mycoses.
For the future, it should be mentioned that nucleic acid sequencing has become the standard method for fungal identification, especially in reference laboratories.
SUMMARY OF FUNGI DIAGNOSIS
|Growth in lesser than 3 Days||Growth 3- 5 days|
|Hyphae are broad and aseptate||Hyphae hyaline and septate|
|Suspect agents of Hyalohyphomycosis Conidia in Chains|
Conidia in Clusters
Gliocladium Conidia Borne Singly
|Growth 3-5 days||Growth > 5 days|
|Colonies often granular and pigmented, hyphae septate, hyaline||Hyphae|
|Suspect Dermatophyte Genus Microsporidia common|
Genus Trichophyton common
|Suspect Dimorphic fungi|
|Growth 3-5 days||Growth > 5 days|
|Dark colony, black reverse, hyphae yellow-pigmented and septate||Smooth, pasty or mucoid colonies|
|Suspect Agent of Phaeohyphomycosis Conidia Muriform|
Conidia divided by Transverse Septa only
Exserohilum Pycnidia produced
|Suspect Agent of Chromomycosis or mycetoma Caldosprium-Type Sporulation Cladophialophora carrunil|
Phialophora – type Sporulation
YEAST AND YEAST-LIKE COLONIES
|YEAST COLONIES||YEAST-LIKE COLONIES|
|Growth 2-5 days||Growth 2-5 days|
|Smooth, pasty or mucoid colonies||Yeast-like colonies with low aerial mycelium|
|Suspect Yeast Common|
Yeast forms of dimorphic fungi
|Arthroconidia Produced Suspect|
Trichosporon beigeliicoraplex Blastoschizomyces capitis