pathology

1. RECENT ADVANCES
IN FUNGAL
INFECTIONS
Speaker : Dr P Agrawal
Guide : Dr B Basu

2. RECENT ADVANCES REGARDING
 Taxonomy and Nomenclature
 Diagnosis of Fungal infections
 Direct microscopic examination
 Culture methods
 Nonculture methods
 Emerging & re-emerging fungal infections
 Unusual & pseudo fungal infections
 Antifungal susceptibility testing
 Antifungal therapy

3. INTRODUCTION
 Term Mycology derived from mykes, a Greek word
for mushroom
 Fungus, a Latin word also means mushroom
 For many years, medical mycology was treated as
“Stepchild”
BUT
 The last 20 years has seen an alarming increase in
fungal infections along with a dramatic increase in
the population of severely ICH
SO
 Now there is an “obligatory” attention to this subject

4. Recent advances regarding Taxonomy
and Nomenclature
 Fungal taxonomy is a dynamic, progressive
discipline requiring frequent changes in
nomenclature
 Sexual stages are the baseline of fungal taxonomy
 Fungal nomenclature is governed by the
International Code of Botanical Nomenclature
[ICBN]
 Taxonomy of some pathogenic fungi are particularly
unstable and controversial at present

5. Recommendations
 Disease nomenclature based on the traditional
structure “fungus+sis” should be discouraged
 Since it is highly influenced by taxonomic
changes
Instead
 Names like “pathology A due to fungus B”
should be encouraged – “ Vaginitis due to C.
albicans”

6. Molecular techniques in phylogeny
 Ribosomal DNA sequencing -
 For filamentous fungi – 18 S sequence
 For yeasts – D1 & D2 variable regions of 25S
 Nuclear DNA G+C content –
 Difference of 1% indicates separate species
 DNA-DNA hybridization & heteroduplex
formation -
 Relative hybridization of 80% - Identity
 Less than 20% - Non identity
 DNA sequencing & electrophoretic methods

7. Nomenclature
 Teleomorph ( sexual state/ perfect state) or
“meiosporic fungus”
 Anamorph ( asexual state/ imperfect state) or
“mitosporic fungus”
 Holomorph – fungi with teleomorphic sporulation
together with all their vegetative anamorphs
 Deuteromycetes – fungi having anamorphs
alone placed in a separate major high level
taxon: changed to lower case “d”
deuteromycetes

8. Lexicon of mycology
 Homothallic- a fungus in which mating can take place
within the same colony
 Heterothallic- a fungus that can only mate between
different colonies
 Hyalohyphomycosis- infection caused by moulds with
light colored colonies
 Phaeohyphomycosis- infection caused by moulds
with dark colored colonies

9. New species
 Coccidioides posadasii within Coccidioides
immitis
 Candida dubliniensis within Candida albicans
Change of name -
 Pneumocystis jirovecii for Pneumocystis carinii
 Zygomycosis for Mucormycosis

10. Diagnosis of fungal diseases
Direct microscopic examination
INDIA INK
PREPARATION
CALCOFLUOR WHITE
STAIN

11. Direct microscopic examination………..
GROCCOTTS STAINFLUORESCENT ANTIBODY
STAIN

12. Direct microscopic examination……….
MASSON FONTANA STAIN PERIODIC ACID SCHIFF

13. Diagnosis of fungal diseases
Culture Methods
 Culture media-
 CHROMagar
 Modifications of Sabauraud agar
 Aspergillus differential medium
 Trichophyton agar 1-7
 Mycobiotic agar/ Mycosel agar
 Bismuth sulfite glucose glycine agar ( BiGGY)
 Inhibitory mold agar
 Kelley’s medium

14. CHROMagar
 Commercially available,
differential and selective
medium used for isolation
& differentiation of
clinically important yeasts
 Contents -
 Peptone
 Glucose
 Chloramphenicol
 Proprietary chromogenic
mixture

15. Findings on CHROMagar
C.albicans Light blue-green
C.dubliniensis Dark green
C.glabrata Pink, white, purple
C.tropicalis Blue-purple with pale pink halo
C.parapsilosis Cream - white
C.krusei Lavender to paler pink
Trichosporon Dirty pink
Geotrichum Pale colony with green halo

16. Recent Yeast Identification Kits
 API 20C system
 Uni-Yeast-Tek system
 VITEK yeast biochemical card (YBC)
 Rapid yeast plus system
 Bactcard Candida Test
 Murex C.albicans Test

17. Bactcard Candida Test
L-proline- β naphthylamide
Proline aminopeptidase
Naphthalene
Red color
Positive
4-methyl umbelliferyl-Nacetyl-β
D-galactosamine
β galatosaminidase
4-methyl-umbelliferone
Bright blue fluorescence
Positive

18. Bactcard Candida Test
PRO MUGAL
2. Inoculate & incubate at RT for 5 min
1. Rehydrate the
circle with reagent

19. Diagnosis of fungal diseases
Nonculture Methods
 Nonculture methods are useful for invasive fungal
infections when neither microscopic nor cultural proof
of infection is available
 Detection of specific host antibodies
 Detection of fungal antigens
 Detection of distinctive fungal metabolites
 Detection of specific nucleic acids

20. Detection of specific host antibodies
 Blastomycosis- Blastomyces dermatitidis
 Antibodies to A antigen
 Antibodies to WI-1 antigen - more reactive & specific
 Coccidioidomycosis- Coccidioides immitis
 Antibodies to tube precipitin (TP) antigen- IgM
 Antibodies to complement fixation (CF) antigen- IgG
 Histoplasmosis- Histoplasma capsulatum
 Antibodies to histoplasmin antigen
 Antibodies to recombinant H antigen

21. Limitations of Ab detection
 Low sensitivity and specificity in invasive candidiasis,
aspergillosis & cryptococcosis
 Poor antibody response in severely ICH
 Cross reactions among different fungal species
 Presence of antibodies to common environmental &
commensal fungi
 Lack of standardization of antigens and methods of
antibody detection

22. Recent advances in Ab detection
 Crude, unfractionated mixture of antigens leads to major
problem of cross reactivity
 More refined antigens using recombinant technology
 More advanced assay systems

23. Composite test for four fungi
 Fungal immunodiffusion system - based on
Ouchterlony double diffusion principle
 Provided with antigens of -
 H.capsulatum
 B. dermatitidis
 C. immitis
 A. fumigatus
 Well 7- for Ag
 Well 2,3,5,6 for pt’s serum
7
1 C 2
3
4 C5
6

24. Detection of Fungal antigens
 Criterion for Ideal antigenic marker-
 Should not be present too transiently
 Should be associated with infection rather than
colonization
 Conserved within the fungal species of interest
 Should not cross react with other human and microbial
antigens
 Should be present early enough to start therapy.

25. Merits of antigen detection
 Useful in diseases where cultures are seldom positive
 Useful when antibody response is poor
Demerits of antigen detection
 Transient presence of antigen due to formation of
immune complexes & removal from circulation
 Use of polyclonal antibodies resulting in variability from
batch to batch

26. Detection of Fungal antigens……..
Invasive Aspergillosis –
Detection of Galactomannan in body fluids
 EIA, RIA, Latex agglutination - (5-15 ng /ml)
Immune complex must be dissociated
Use of more sensitive systems
Use of monoclonal antibodies
 Sandwich ELISA (Platelia Aspergillus) using
rat monoclonal Ab EB-A2 (0.5-1.0 ng /ml)
TO IMPROVE
SENSITIVITY

27. Detection of Fungal antigens……..
Invasive Candidiasis
 Cand-Tec latex agglutination test (unknown Ag)
 Enolase
 β-(1-3)-D-glucan (Fungitec G-test)
 Mannan detection -
 Latex agglutination (Pastorex Candida)
 Sandwich ELISA (Platelia-Candida) using monoclonal
antibodies EB-CA1
 To improve the sensitivity, repeat sampling or
combination of two assays is recommended

28. Detection of Fungal antigens……..
Cryptococcosis
Cryptococcal capsular polysaccharide antigen
 Latex agglutination tests-
 Polyclonal IgG based
 Monoclonal IgM based
 CALAS - Pronase
 PREMIER EIA – advantages over LAT
 Does not react with RA factor
 More sensitive
 Rapid

29. Detection of distinctive fungal
metabolites
 Specific fungal metabolic products can be demonstrated
in the body fluids of infected host
 Criterion for diagnostic metabolic marker
 It should be produced in large amounts in culture & in
the infected tissue
 It should be present in trace amounts in uninfected
control
 method of detection should be sensitive and accurate

30. Detection of distinctive fungal
metabolites………….
 Ethanol in CSF samples in C.neoformans meningitis
 Oxalic acid crystals in lungs in Aspergillus fungal ball
 Polyol mannitol in aspergillus and cryptococcus
infection
 D-arabinitol in invasive candidiasis
Methods of detection-
 Gas chromatography (GC)
 Gas chromatography with mass spectrometry (GC-MS)
 Enzymatic chromogenic assay

31. Detection of specific nucleic acids
 Target selection
 Single and multicopy nuclear and mitochondrial genes
are used
 Conserved sequences - for screening purpose
 Variable sequences – for species identification
 Methods used
 Single PCR
 Nested PCR
 PCR-EIA
 Real -Time PCR( Light Cycler by Roche Diag)

32. Detection of specific nucleic
acids…….
MERITS-
 Rapid
 Accurate
 High sensitivity and specificity
 Ideal for slow growing/difficult to culture fungi
DEMERITS-
 CANNOT distinguish between colonized host and
infected host

33. Overview of fungal diagnostic
methodology in IFI
 Traditional culture and morphological methods
may be insensitive or nonspecific but they are
the best methods in routine clinical lab
 A multimodality approach is recommended by
combining the traditional with one or more of the
newer diagnostic methods

34. Emerging & Re-emerging fungal
infections
 Zygomycosis – due to increased use of
voriconazole prophylaxis
 Increase in non-albicans Candida infections
 Chronic disseminated Candidiasis
 Cryptococcosis and late onset invasive
Aspergillosis (IA) in SOT recepients

35. Unusual fungal infections
PULMONARY
ADIASPIROMYCOSIS
Emmonsia crescens

36. Unusual fungal infections
LOBOMYCOSIS
Lacazia loboi

37. Pseudo fungal infection
CHLORELLOSIS
Chlorella sp.

38. Pseudo fungal infection
RHINOSPORIDIOSIS
Rhinosporidium seeberi

39. Recent advances in antifungal agents
 Polyenes - Amphotericin B
 Lipid associated formulations -
 Am B colloidal dispersion
 Am B lipid complex
 Liposomal Am B
 Azoles- Imidazoles & triazoles
 Voriconazole
 Posaconazole
 Ravuconazole

40. Recent advances in antifungal
agents………..
 Echinocandins –
 Anidulafungin
 Caspofungin
 Micafungin
 Antimetabolites -
 Flucytosine (5FC)
 Chitin synthase inhibitors –
 Nikkomycin

41. Resistance to antifungal drugs
There are recent reports of -
 Azole resistant C. albicans among HIV
seropositive men & women
 Multi-Echinocandin-Resistant C. parapsilosis
among burn patients who remain susceptible to
fluconazole and voriconazole

42. Recent advances in antifungal
susceptibility testing
 Standard guidelines –
 NCCLS M27 A document for yeasts &
 NCCLS M38 A document for moulds
 Standardized Methods –
 Broth macrodilution
 Broth microdilution – more widely accepted
 Principle –
 Visual comparison of turbidity of test fungi with turbidity
of 80% inhibition of standard

43. Recent advances in antifungal
susceptibility testing……..
 Media-
 RPMI 1640 broth with 0.2% dextrose & buffer MOPS
( pH 7.0)
 Yeast nitrogen base broth with MOPS for better growth
of C. neoformans
 RPMI 1640 broth with 2% dextrose
 Quality Control-
 C. parapsilosis – ATCC 22019
 C. krusei – ATCC 6258

44. Alternatives
 Broth based -
 Colorimetric method
 Spectrophotometric method
 Break point testing
 Flow cytometry
 Agar based –
 Epsilometer strip test (E-Test)
 Disk diffusion test

45. Recent advances in antifungal therapy
Combination therapy
 For Candida spp-
 Caspofungin + Am B
 Caspofungin + 5-FC
 Voriconazole + micafungin
 For Cryptococcus-
 5FC+ Fluconazole/ Am B
 Voriconazole + Caspofungin
 For Aspergillosis-
 Voriconazole + LAmB / Caspofungin
 Caspofungin + 5-FC

46. Miscellaneous combinations
 Combinations of Antifungal, Antibacterial &
Anticancer agents
 Ciprofloxacin + Am B/ Flu
 Pentamidine + Itra / Keto
 5FC + AmB/ Flu
 Antifungal combined with immunomodulators
(G-CSF,GM-CSF ,PMN’s)
 AmB lipid complex + PMN’s
 LAmB + G-CSF

47. REFERENCES
 Mandell – Principles and Practice of Infectious diseases ;
6th
ed, chapter 37 & 254
 Murray – Manual of Clinical Microbiology; 8th
ed, chapter
110-124
 Koneman – Color Atlas and Textbook of Diagnostic
Microbiology, 5th
ed, chapter 19
 45th
Annual Interscience Conference on Antimicrobial
Agents and Chemotherapy, Clinical Mycology and
Antifungal Therapy CME ;www.medscape.com

48.  Developments in fungal taxonomy; Clin. Microbiol
Reviews, July 1999, p 454-500
 Current Status of Nonculture Methods for Diagnosis of
Invasive fungal infections; Clin. Microbiol Reviews,
July 2002, p 465-484
 Combination treatment of invasive fungal infections;
Clin. Microbiol Reviews, Jan. 2005, p 163-194
 Minireview;Unusual fungal & pseudossfungal infections
of humans; J. Clin. Microbiol, Apr 2005, p1495-1504

49. Thank You