• Proper diagnosis of the infections aids in the treatment procedure. So, the combination of clinical observations along with laboratory investigations is crucial for the diagnosis of the infection.
• Different diagnostics approach applied to fungal infections are:
  1. Clinical approach for diagnosis of fungal disease
  2. Conventional Methods for diagnosis of fungal disease
  3. Molecular Methods for diagnosis of fungal disease
  4. Recently developed new techniques for diagnosis of fungal disease
  5. Other Miscellaneous Methods

Clinical approaches for diagnosis of fungal disease:

• The characteristics of lesions produced by superficial and subcutaneous mycoses suggest their fungal etiology though it may resemble other diseases too.
• There is no particular sign and symptom in the case of systemic mycoses. The infection is similar to bacterial, viral, or parasitic disease.
• Modern imaging techniques aids in the early diagnosis of fungal disease.
• Based on it and clinical significance, the suspected disease can be further identified by laboratory investigations.

Conventional (microbiological) approaches for diagnosis of fungal disease: 

Based on Sites and Types of Specimen:

• For the proper diagnosis of fungal disease, the specimen type and the site of collection should be accurate.
• a) Superficial Mycoses:
  • The site should be cleaned using 70% alcohol. Before taking the specimen it should be allowed to evaporate.
  • The material should be collected at the folded square of paper because it:
  • Permits drying of the specimen
  • Reduces bacterial contamination
  • Stores for a long time without losing the viability of fungi
  • Dermatophytic lesions should be collected using the scalpel blade which is held at 90°C to the skin surface. From the edges of the active lesion, materials should be scrapped outward and collected.
  • In case of lesions on glabrous skin ( smooth or hairless), cellophane tape can be used.
  • Scalp specimen should be collected with blunt scalpel including hair-stubs, contents of plugged follicles, and scales.
  • Instead of cutting, hair should be plucked.
  • Ringworm infection of the scalp is detected by Wood’s lamp examination. The infection hair produces fluorescence.
  • To obtain a specimen for fungal culture, a hairbrush sampling technique can be used. In this technique, the scalp is brushed thoroughly which is then used for inoculation on culture media.
  • Patients should be off antifungal agents, one week before the collection of specimens in the case of onychomycosis ( fungal infection of nails ).
  • The nail should be disinfected with 70 % alcohol and clipped from the free edge.
  • Culture can also be done from the borings taken from the base of the nail.
  • Scrapings are preferred over the swab for the infection of the mucous membranes.
• b) Subcutaneous Mycoses:
  • For the microscopy and culture, scrapings taken from the superficial parts of the subcutaneous lesions may be satisfactory.
  • Contamination is common with bacteria and saprophytic fungi. The aspirated sample of pus and/or biopsy material is valuable.
  • If sporotrichosis is suspected then a biopsy may be avoided. It is because of the chances of spread of infection and the lesions will also be healed slowly.
• c) Systemic Mycoses:
  • Specimen: Biopsy, pus, feces, urine, sputum, spinal fluid, blood, scrapings, or swabs from the edge of lesions.
  • The urine taken from the catheter bag and twenty-four hours’ sputum is unsatisfactory. It is because the commensal yeast can multiply rapidly.

Collection and transport of different fungal specimens

• a) Respiratory specimens:
  • Specimens: sputum, tracheal secretions, bronchoalveolar lavage (BAL), lung biopsy
  • An early morning sputum sample is collected.
  • In case of non-productive cough, sputum may be induced
  • Bronchoscopy is also used for the examination of lesions and collection of specimens in respiratory mycoses.
  • For concentrating the specimens:
    • Add 0.5 gm of N-acetyl L-cysteine (NALC) in sodium citrate buffer which is prepared freshly.
    • Then vortex for 10-30 seconds
    • Then M/15 phosphate buffer with pH 7.0 is added. Its volume should be double the volume that is already present in the tube.
    • Then centrifuge at 1000 rpm for 15 minutes.
    • The supernatant is discarded and the sediment is used for smear preparation and in media inoculations.
  • Opportunistic pathogens like Candida can be present in the sputum as the oropharyngeal contaminant. The presence of Candida in the respiratory specimens is not clinically significant unless it is found in tissue. Bronchial brushing and lung biopsy may also be used.
• b) Cerebrospinal Fluid:
  • For the culture of CSF, it should be centrifuged and the sediment is inoculated in the agar.
  • Inhibitory agents should not be used in the media because, in normal conditions, CSF is always sterile.
  • CSF should not be processed immediately. It should be kept at room temperature or incubated at 30°C
• c) Blood Culture:
  • Biphasic Brain-Heart Infusion Agar Broth can be used.
  • Though most of the fungi can be detected within the first four days of incubation, an occasional isolate of Histoplasma capsulatum may require 10 to 14 days.
  • Blood culture media should be incubated at both the 25°C and 37°C temperature.
  • Subculture should be done at two days and seven days respectively.
  • After the seven days of subculture preliminary report and after 28 days of subculture final report is sent.
• d) Tissue, Bone Marrow, and Body Fluids:
  • From the pyogenic and the necrotic areas of the wounds, tissue specimens should be taken.
  • Povidone-iodine should not be used as the chances of isolation of fungi will be bleak. It may be applied after the collection
  • The tissue specimen should be minced before culture. Then it should be inoculated in appropriate culture media and incubated at 37°C for 4 weeks.
  • Bone marrow can be directly inoculated on media and incubated.
  • Before the culture, centrifugation should be done for the body fluid which is collected from a sterile site.
• e) Semen Culture:
  • Done in Histoplasmosis and Cryptococcosis when the disease is somewhat hidden and patient without any improvement has taken repeated courses of antitubercular treatment.
• f) Skin:
  • For dermatophyte infection, skin scrapping should be used.
• g) Nail:
  • The nail should be clipped from the discolored, dystrophic, or brittle parts.
  • Since, in the distal part of the nail, the fungus is non-viable, it fails to grow in culture.
  • Before the inoculation in the suitable culture media, nails should be cut into pieces.
  • It can be only visualized by microscopy.
• h) Hair:
  • Instead of cutting, hair needs to be plucked with forceps.
  • It is then kept in a sterile petri dish or paper envelope.
  • Since low temperature may be detrimental to the dermatophytes, it should not be refrigerated.
  • Dermatophytes are cultured in Sabouraud dextrose agar with chloramphenicol and cycloheximide.
  • Before reporting them as sterile, cultures should be incubated at 25°C, 30°C, and 37°C for a minimum period of four weeks.
• i) Urine culture:
  • Twenty-four hour’s urine is not useful for the fungal culture.
  • If a delay is anticipated, it should be refrigerated at 4°C. It can be kept for up to 12 hours.
  • Before the culture, the urine sample needs to be centrifuged and the sediment is inoculated.
  • Culture media should contain antibacterial antibiotics to prevent bacterial contamination and isolate the fungi in pure form.
• j) Vaginal Secretions:
  • Clinical features along with the direct smear of secretions help in the diagnosis of vaginal candidiasis.
  • About 20 % of healthy females have yeasts as the normal flora, so culture can be misleading too.
  • Cultures can help monitor the therapy and for the management of chronic recurring diseases.
• k) Stool culture:
  • Biopsy of tissue is done than the culture of stool specimens for the diagnosis of fungal infections in the gastrointestinal tract.
  • Since yeast colonizes as the commensals in 40% of healthy individuals and 75% of compromised patients, positive cultures may be misleading.
• l) Eye:
  • Corneal scrapings are taken in the case of keratomycosis.
  • Kimura’s spatula is used aseptically to take the sample from the base and margin of the ulcer.
  • 4% xylocaine is used as the local anesthetic.
  • In keratomycosis, aspiration of hypopyon is done using the sterile needle.
  • In the case of fungal endophthalmitis, the posterior chamber may also be aspirated.

Sample preparation for laboratory diagnosis of Fugal disease

a) Direct Microscopic examination:

• The direct demonstration of fungi in the clinical specimen is taken as the “gold mine”.
• Fungi can be observed directly in the clinical specimens by:
  • Wet Mounts
  • Histopathology
  • Frozen-Section Biopsy
  • Fluorescent-Antibody staining

i)  For Wet Mounts preparation:

• Slide and tube KOH Mounts are prepared.
• Sodium hydroxide may also be used as an alternative.
• After the partial digestion with 10-20% KOH, specimens can be examined in wet mounts.
• On the slide, specimens like hair, nail, skin are mounted in KOH under the coverslip.
• Materials are digested under 5-20 minutes depending on the thickness.
• Under the low flame, it can be lightly warmed but should not be overheated.
• DMSO can be supplement in KOH to increase clearing of the fungi in the skin scrapings.
• Calcofluor and Blankophor are also used to prepare wet mounts. It offers excellent visualization of the fungi.
• The fungal cell wall under ultraviolet illumination, fluoresce brightly under the fluorescence microscope.
• For the detection of Cryptococcus wet mounts of India ink and Nigrosin staining are used.
• For evaluation of the viability of fungi, neutral red staining can be used.
• For the demonstration of fungi like Malassezia , Candida spp. and dermatophytes, Vinyl Adhesive Tape (VAT) preparation is also used.

ii) For Histopathology:

• Demonstration of the fungi in the tissue sections aids in diagnosis.
• If histopathology shows neither the fungal elements nor the tissue reaction, the fungal isolate can be the contaminant.
• Histopathological examination of the biopsy and autopsy specimens is the best method for the diagnosis of mycotic infections.
• In the histopathology laboratory, H&E stain is a routine procedure.
• For demonstrating fungi in tissue-specific fungal stains, such as Periodic Acid-Schiff (PAS), Grocott-Gomori’s methenamine silver stain, and Gridley stains are widely used.
• For a demonstration of capsular material of Cryptococcus and endospores and sporangia of Rhinosporidium seeberi, Mayer’s mucicarmine can be used.
• To demonstrate acid mucin, Alcian blue staining may be done.
• Disadvantage of use of special stain:
• Mask the natural color of fungal elements making it difficult to decide if it is hyaline or naturally pigmented.

iii) Frozen-Section Biopsy:

• This modality is adopted for making an intra-operative diagnosis of suspected malignancy.
• In mucormycosis and fungal rhinosinusitis, good results have been obtained.
• It is a useful tool to guide the extent of surgical debridement and/or onset of antifungal therapy.
• Frozen sections describe the morphology and infectious process.
• The evaluation of the frozen section has an important function in surgical pathology for diagnosis in tissue while the patient is undergoing an operative procedure.

iv) Fluorescent-Antibody Staining:

• It is used for the detection of fungal antigen in clinical material such as pus, blood, CSF, tissue impression smears, and in paraffin sections of formalin-fixed tissue.
• For the sputum specimen, it is less satisfactory.
• Advantage: detects fungus when few organisms are present.

b) Fungal Culture:

• Commonly employed medium is Emmons’ modification of Sabouraud Dextrose Agar.
• To minimize bacterial contamination, gentamicin and chloramphenicol can be supplemented in the media.
• To inhibit the saprotrophic fungi, cycloheximide can be supplemented in the media.
• Some fungi like Cryptococcus, Talaromyces marneffei, Aspergillus, or Scytalidium are sensitive to cycloheximide. So, cycloheximide should not be used in this case.
• For the isolation of the particular pathogens, a special medial can be used.
• For isolation of neoformans, Birdseed agar, Niger seed agar, Sunflower seed agar can be used.
• neoformans develop brown-colored colonies.
• Candida , Aspergillus spp. , Rhizopus spp. grows within 24-72 hrs of incubation.
• For the dimorphic fungi, incubation should be done at:
• 37°C for the isolation of yeast.
• Room temperature for the isolation of molds.

Techniques for detection of fungi from culture:

• • Scotch Tape preparation
  • Wet Mount preparation
  • Lactophenol cotton blue staining
  • Germ Tube production test

i. Scotch Tape preparation:

• Over the growth of filamentous fungi, the sticking surface of scotch tape is touched.
• Then it is placed on a glass slide and observed under the microscope.
• Characteristics shape and arrangement of spores, type of hyphae and conidia, etc can be observed.
• Advantages:
  • It can be prepared quickly and easily.
  • Slide can be preserved for a longer time.
  • Fungi can be seen with their own pigmentation.
• Disadvantages:
  • Sample won’t be adequate if it is not pressed firmly.

ii. Wet Mount preparation:

• It is done for the observation of spores that were not seen by scotch tape.
• Example: Microconidia of Histoplasma capsulatum can be observed by the wet mount method.
• Procedure:
  • Bent wire of 90° is used.
  • Small portion is cut at the intermediate point of the center and periphery of the isolated colony.
  • It should contain a small amount of the supporting agar.
  • Then a drop of KOH is added.
  • It is covered by a coverslip and observed under the microscope.
• Disadvantages:
  • If pressure is applied to the coverslip, the characteristic arrangement of spores will be disrupted. In such a condition, definitive identification of fungi can’t be done.

iii. Lactophenol cotton blue staining:

• Fungal cytoplasm can be stained against which the walls of hyphae can be readily seen.

iv. Germ Tube production test:

• It is used for the definitive identification of fungi within 3hrs.
• Example: Yeast, Candida albicans
• Germ tube is the elongated tube-like structure originating from yeast cells.
• Nucleus is absent.
• It has half the width and 3-4 times greater the length of the yeast cell.
• Procedure:
  • Suspend the inoculums from the isolated colony of yeast cells in 0.5 ml sheep or rabbit serum.
  • Incubate the tube at 37°C for 3-4 hrs.
  • Then take the suspension and observe under the microscope.

c) Serological test:

• Agglutination tests:
  • Whole-cell agglutination (WCA)
  • Latex particle agglutination (LPA)
• Passive haemagglutination (PHA)
• Immunodiffusion (ID)
• Counterimmunoelectrophoresis (CIE)
• Complement fixation (CF)
• Indirect fluorescent antibody (IFA)
• Enzyme-linked immunosorbent assay (ELISA)
• Radioimmunoassay:
  • Solid-phase
  • Competitive RIA
• Complement fixation test (CFT):
  • Coccidioidomycosis, Histoplasmosis, Blastomycosis
  • Presence of the polysaccharide capsular antigens of  Cryptococcus neoformans in the CSF can be detected by the latex agglutination test.

Skin Test for fungal disease:

• Individuals infected with the Histoplasma or Coccidioides develop a Delayed-Type-Hypersensitivity (DTH) reaction with in1-14 days and may persist for many years
• Appropriate fungal antigen is inoculated intradermally.
• Within 24-72 hours induration and erythema occur.
• Skin test is used for:
• Establishing the etiological diagnosis
• Conducting the epidemiological survey
• Immunological classification of the subjects like atopic and non-atopic groups
• Find out the immunological status of the patients as in the immunodeficiency diseases

Various skin tests and fungal antigens used:

Molecular approaches for diagnosis of fungal disease:

1. Hybridization methods
2. Amplification methods
   • Broad range PCR
   • Nested PCR
   • Multiplex PCR
   • Nucleic acid Sequence-based Amplificatio
   • Fluorescence Resonance Energy Transfer
   • TaqMan
   • Molecular Beacons

3. Sequencing-based methods
   • Sanger’s Sequencing
   • Pyrosequencin
   • Next-Generation Sequencing
   • Ultra-Deep Sequencing
   • DNA Bar Coding

Recently Developed new Techniques for diagnosis of fungal disease:

• AccuProbe
• PNA FISH
• MALDI-TOF Mass Spectrometry
• Quantamatrix Multiplexed Assay Platform

Miscellaneous Methods:

• Point of Care Testing
• T2Candida Panel
• Biosensors for Medical Mycology
• Epidemiological Markers of Fungi
• Quality Control in Medical Mycology

Epidemiological Markers Used in Fungal Infections:

• Phage typing
• Secreted lethal factor typing
• Serotyping
• Morpho typing
• Mating typing
• Resistotyping
• Biotyping
• Protein electrophoresis (Immunoblot)
• Isoenzyme typing
• Restriction fragment length polymorphism (RFLP)
• Karyotyping
• Nucleic acid probes

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