This document summarizes various laboratory methods for the diagnosis of fungal infections. It discusses sample collection and storage, direct microscopic examination of samples, fungal culture techniques, identification of fungal isolates, and serological diagnostic methods like antigen and antibody detection. Key points covered include the use of KOH, calcofluor white, and PAS stains for direct examination of samples, as well as the growth of fungi on sabouraud dextrose agar and brain heart infusion agar for isolation and identification based on colony characteristics and microscopy. Serological tests mentioned include agglutination, immunodiffusion, and ELISA.

1. Laboratory Diagnosis
of Fungal Infections

2. Classification
Based on molecular evidence: base sequences from ribosomal RNA (Patterson & Sogi

3. Comparison of fungi and bacteria
Feature fungi bacteria
Diameter 4um 1um
Nucleus Eukaryotic prokaryotic
Cytoplasm Mitochondria and
endoplasmic reticulum
present
Mitochondria and
endoplasmic reticulum
absent
Cell membrane Sterols present Sterols absent
Cell wall chitin peptidoglycan
Spores Sexual and asexual spores for
reproduction
Endospores for survival,
not for reproduction
Thermal
dimorphism
yes No
Metabolism Require organic carbon; no
obligate anaerobes
May do not require
organic carbon; many
obligate anaerobes

4. It's advisable to collect as much material as
possible due to scarcity of many fungi in many
specimens.
The type of the sample collected is determined
according to the suspected site of infection
such as:-skin scrapping – nail clipping – hair
epilation – sputum – blood, C.S.F.
Sample

7. Sampling using blunt scalpel

8. Specimen Collection

9. Sampling using cotton swab

10. Sampling using Biopsy

11. Skin, nail and hair:
They are collected in air-tight containers such as
small glass bottles.
The fungi in test samples can remain viable for
weeks or even months.
They shouldn't be refrigerated as the viability of
some species of dermatophytes is affected.
Storage and transport

12. Other specimens should be collected, stored
and transported in a manner similar to that
employed for bacteriological investigation.
If specific fungus is suspected, the lab should
be notified as special media and culture
procedures may be needed and that also will
be helpful for the safety of lab personnel.

13. Laboratory Diagnosis
Direct examination: Very decisive in the
diagnosis of fungal infections
Fungal culture
Serological tests
Skin tests
PCR & other molecular methods

14.  Give an early indication of the presence of yeast especially if
from normally sterile tissue provided the sample collected
aseptically,
 The presence of hyphea helps in establishing differential
diagnosis.
 The observation of capsule surrounding yeast gives a
presumptive diagnosis of cryptococcosis.
 It provides physician with early information regarding the
possible need of treatment.
 Guide the microbiologist about the type of culture media used.
Direct examination

15. A) Unstained preparations (wet mount) :
KOH preparation for digestion of organic substances
and keratinized material giving a clear background
without affecting the fungus. DMSO can be added to
KOH to hasten clearing in skin scrapings & nail
clippings.
India ink preparation to detect capsulated fungi i.e:
Cryptococcous neoformans.
Methods

16. Direct Examination

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CFW – yeast form of
Blastomyces
KOH - Aspergillus India ink -
Cryptococcus

18. B) Stained preparations :
 lactophenol cotton blue.
 Calcoflour white fluorescent stain.
 Gram stain for detection of aerobic actinomycetes.
 Modified kinyoun acid fast stain for detected of partially acid
fast filaments of Nocardia species.
 Wright's stain and Giemsa stain to detect intracellular yeast
forms of Histoplasma capsulatum in blood and bone marrow.
 PAS and Gomori's methanamine silver stain for demonstrating
fungi in tissue.

19.  Fluorescent- antibody staining
 To detect fungal Ag in clinical specimen such as
pus, blood, CSF, tissue sections
 Adv – can detect fungus even when few
organisms are present
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20.  Gram stain – fungi are Gram positive

32. PAS

33. GMS

34. H&E

35. Mucicarmine

36.  Sabouraud Dextrose Agar (SDA)
 Cyclohexamide should be added to sabauraud's for isolation of primary
pathogens and inhibit opportunistic pathogens.
 Chloramphenicol or gentamicin to inhibit bacterial contamination.
 Selective media
 Brain Heart Infusion (BHI) agar :dimorphic & other fastidious fungi
Fungal Culture

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Corn Meal Agar
Bird Seed Agar

38.  All fungi are aerobic
 Temperature requirement : temperature:25-27°C (for filamentous
fungi) or at 37°C (for yeast).
 Majority of fungi – 37°C
 Superficial mycosis – 30°C
 Dimorphic fungi – 25°C & 37°C
 Incubation time:,
 At least 4 weeks
 Usually positive cultures are obtained in 7-10 days
 Candida & Aspergillus - 24 to 72 hrs

39.  Specimens should be cultured on agar slants:
 Safe
 Require less space
 More resistant to drying during prolonged
incubation
 Blood cultures should be inoculated in to biphasic
blood culture bottles

40. Interpretation of Fungal Culture
 Isolation of an established pathogen like H. capsulatum or
C. neoformans ; evidence of infection
 Isolation of commensal or opportunistic fungi like
Candida or Aspergillus ; consider following points:
1. Isolation of same strain in all culture tubes
2. Repeated isolation of same strain in multiple specimens
3. Isolation of same strain from different sites
4. Immune status
5. Serological evidence

41. Identification of fungal cultures
Colony character: Macroscopic examination of isolated
colonies for the texture aspect, colour of the colony
surface and the reverse of it, any pigment that diffuse in
the medium.

42. Microscopic examination:
Fungal morphology under
microscope using
Lactophenol Cotton Blue
(LPCB) stain
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43. Biochemical reactions :
Fermentation of sugars
Assimilation of sugars.
Assimilation of N2.
Other tests as urease test.

44. Identification of Candida albicans:
 The colonies are rounded white creamy in colour and creamy in
texture.
 Film stained by lactophenol blue show budding yeast and
filaments (pseudo-mycelium).
 If stained by Gram stain appears Gram positive.
 Germ tube test: culture of the yeast on serum causes rapid
formation of filaments when incubated at 37°C for 4 hours.
 Fermentation of sugars.

45. Serology
Detection of Ag or Ab in serum or body fluids
Ab detection:
Diagnosis of systemic & subcutaneous
mycoses
Assess prognosis of the disease
Assess response to treatment
Ag detection:
Early stages of infection
In patients with impaired immunity
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46. Experimental Animal infection
Experimental animal inoculation is sometimes
useful for the isolation of the causative fungus
especially in deep mycoses..

47. Mouse

48. Guinea Pig

49. Guinea pig & Rabbit

50. Hamster

51. Serological tests used in Medical
Mycology
 Agglutination
 Immunodiffusion – most widely used
 Counter immunoelectrophoresis (CIEP)
 Indirect fluorescent Ab detection
 ELISA, RIA
 Delayed hypersensitivity reaction (Fungal skin test) :
It has no value in diagnosis. Mainly used for epidemiological study

52. Other Methods
Nucleic acid detection:
PCR
RFLP
Protein electrophoresis
DNA hybridization (Nucleic acid probes)
Woods light: it is an UVR produce fluorecent color
when come in contact with fungal infection of skin
and hair

53. Thank You