Overview of medical mycology

1. OVERVIEW OF MEDICAL
MYCOLOGY
Dr. Dinesh Jain
Assistant Professor
Deptt. of Microbiology
SMS MC Jaipur

2. INTRODUCTION
 Medical Mycology is the study of fungi
that have an impact on human health
in some form
 increased importance over the years
, -increase in the use of
immunosuppressive drugs
- increase in the incidence of
opportunistic fungal infection

3. Introduction...
 Thus focus of medical mycology has
shifted from a science dealing
exclusively with medical
dermatological conditions to one that
also deals with systemic diseases
leading to considerable morbidity and
mortality

4. PRESENT RESEARCH
 Research aimed in several directions :
- Improved diagnostic testing
- Serological testing
- Molecular Biology
- Search for newer antifungals
-Understanding the mechanisms of
fungal pathogenicity

5. Morphology
 Two basic growth forms or stages :
1. YEAST – Unicellular
2. FILAMENTOUS OR MOLD FORM

6. CLASSIFICATION
 About 1.5 million species
 About 300 species cause human
infections
 About 30 species most commonly
isolated
 Based on teleomorph state, 3 main phyla
-
PRIMARY – ZYGOMYCOTA
- ASCOMYCOTA
- BASIDIOMYCOTA
deuteromyces (artificial class)– 4th fungi
without perfect state, representing asexual
states or anamorphs of Basidiomycota or

7. MEDICALLY IMPORTANT
FUNGI
 ZYGOMYCOTA
 Subcutaneous zygomycosis
 oppurtunistic fungal infections
Entomophthorales and Mucorales
1. Conidiobolus, Basidiobolus
2. Mucorales – Rhizopus, Mucor,
Rhizomucor, Absidia, Cunninghamella,
Mortirella, Saksenaea,
Apophysomyces

8. MEDICALLY IMPORTANT FUNGI
(contd)
 ASCOMYCOTA (Sac fungi)
Pneumocystis jirovecii ,Fusarium ,
 BASIDIOMYCOTA (club fungi)
- Mushrooms,Schizophyllum commune

9. Deuteromycetes
-Blastomycetes=unicellular yeasts
Saccharomyces ,Candida,Trichosporon
-Coelomycetes=Pycnidia(asexual spores);
Phoma-produces skin lesions , corneal ulcers
Hyphomycetes=most important group causing
superficial to systemic infections.eg- Dermatophytes,
Aspergillus,Blastomyces,Histoplasma

10. Classification of Mycoses
According to tissue involved,
MYCOSES
Superficial Deep
Surface Cutaneous Subcutaneous
Systemic
Primary Systemic
Systemic

11. Surface Mycoses
Affect Stratum corneum/hair
1. PITYRIASIS VERSICOLOR-
- Agent : Malassezia furfur
2. TINEA NIGRA -
- Black or brownish macular lesions especially of
palms
- Agent : Exophiala werneckii
3. PIEDRA
- Hair – irregular nodules along the hair shaft
- Black piedra : Piedraia hortae
- White piedra : Trichosporon beigelii

12. CUTANEOUS MYCOSES
1. DERMATOPHYTOSES
- Infection of keratinized structures like skin, hair,
nails by keratinophilic fungi called dermatophytes
2. DERMATOMYCOSIS
- Skin lesions of Candida
- Cutaneous menifestations of systemic mycoses

13. DERMATOPHYTES
- 3 genera :-
1. Microsporum - M.gypseum, M.canis
2. Trichophyton – T.rubrum, T.mentagrophytes,
T.verrucossum
3. Epidermophyton – E.floccosum
Clinical Classification according to site involved - Tinea
capitis, T. barbae, T. corporis,---

14. DEEP MYCOSES
SUBCUTANEOUS MYCOSES
1. Mycotic Mycetoma
2. Chromomycosis (
Chromoblastomycosis and
Pheohyphomycosis)
3. Rhinosporidiosis
4. Subcutaneous phycomycosis or
Entomorphthoramycosis

15. MYCOTIC MYCETOMA
 Gill (1842)-Madura foot from Madurai in South
India.
 Three types
-Eumycetoma =caused by fungi eg –
Scedosporidium, Madurella mycetomatis,
Acremonium, Exophiala, Aspergillus,---
-Actinomycetoma=caused by filamentous bacteria
eg Actinomadura,Nocardia,--
-Botryomycosis=Staphylococcus aureus
(grains of various colors-
white,yellow,red,brown,black)

16. SYSTEMIC MYCOSIS (DIMORPHIC
FUNGI)
- Soil saprophytes
- Asymptomatic to latent disease
- Thermally dimorphic fungi
- Overcome physiological and cellular
defenses i.e. change their
morphological forms

17. Systemic Mycoses
1. Blastomycosis
- lungs
- Blastomyces dermatidis
2. Paracoccidiodomycosis
- Skin, mucosa, lymph nodes, internal
organs
- P.brasiliensis
3. Coccidiodomycosis
- Primary pulmonary
- Coccidiodes immitis
4. Histoplasmosis
- Intracellular infection of reticuloendothelial
system
- H.capsulatum

18. OPPORTUNISTIC MYCOSES
 Fungi are part of normal commensal flora
of body
 Occur in immunocompromised host
1. Aspergillosis
- A.fumigatus, A.niger, A.flavus
- ABPA, Aspergilloma, Invasive
aspergillosis, Superficial infections of eye
(mycotic keratitis)
& ear (Otomycosis)
2. Penicillosis – P.marneffei (HIV)
3. Zygomycosis – Rhino-orbito-cerebral
form, Mucormycosis, Phycomycosis
……..

19. OPPORTUNISTIC
MYCOSES..
4. Candidosis
- Cutaneous, mucocutaneous,
systemic
5. Cryptococcosis
- C.neoformans
- Pulmonary, visceral, cutaneous,
meningeal
disease
6. Pneumocystis jirovecii - Pneumonia

20. MYCOTIC POISONING
 Mycetism – fungus is eaten itself
- Claviceps, Coprine, Inocybe
poisoning
 Mycotoxicosis – ingestion of food
contaminated with mycotoxins
- Aflatoxins – Aspergillus spp. (groundnuts,
corn, peas)
- Fumonism – Fusarium (maize)
- Ochratoxin – Aspergillus spp. (cereals)
- Penicillium spp. (bread)
- Ergot alkaloids – toxic alkaloids
- Claviceps (rye)
- Fusarium -

21. Present scenario
 India has one of the highest rates of
Candida bloodstream infection in the
world , Article :Chakrabarti
Candidaemia in ICU in India ; Press
release (February 17 2015)
 6.51 cases per 1,000 ICU admissions
seen
 Mortality varied from 35-75%;

22. Emerging fungal infections
 Emerging epidemiological trends in
immunocompromised increased incidence of
invasive mold infections such as Aspergillosis.
 The emerging invasive fungal infections in the solid-
organ transplant recipients are candidiasis (53% of
all invasive fungal infections found) followed by
invasive aspergillosis (19%), cryptococcosis (8%),
non-Aspergillus molds (8%), endemic fungi (5%),
and zygomycosis (2%) [8]. There is increase in
incidence of infections caused by nonalbicans
Candida like C.tropicalis,C.parapsilosis,..
 Our study C.tropicalis highest incidence,(33.33%)
(Journal of Bacteriology & Mycology,Volume 1 Issue 2
- 2015 Ravikant, Tanveer Kaur, Satish Gupte* and
Mandeep Kaur Department of Microbiology, Gian

23. Emerging fungal infections..
 The emergence of zygomycosis
warrants further discussion,
 Zygomycetes, Fusarium,
Scedosparium, Paceilomyces,
Trichoderma, Scopulariopsis,
Dematiaceous fungi (Exophilia,
Alternaria, and
Bipolaris),Chromoblastomycosis,
Trichosporon, Malassezia,
Rhodotorula, Penicillum marneffei,
Paracoccidioides and Sporothrix are
emerging in immunocompromised

24. Common fungal pathogens in
HIV
 Candida albicans
 Cryptococcus neoformans
 Coccidiodes immitis
 Blastomyces dermatidis
 Aspergillus fumigatus
(Ravikant et al)

25. Emerging fungal infections
among children
 Oral thrush - Candida albicans
 Candida diaper rash
 Tinea infection (ringworm)
 Subcutaneous fungal infections- Sporotrichosis
,chromoblastomycoses
 Systemic infections
 Oppurtunistic infections
 Our lab –Candiduria
 Akansha Jain, et al Journal of Pharmacy and Bioallied
Sciences October-December 2010 Vol 2 Issue

26. Our experience at SMS
 Study on cutaneous mycoses (Vyas et
al-2012)
 Culture positive-37.5%, 66.6% were
dermatophytes
 Tinea capitis in 50% cases
 T.violaceum in 32.5% most common
isolate
 Candidimea in ICU-4.32% cases in
ICUs of SMS hospital (K. l.
Verma,A.Vyas,..May,2016)

27. SMS….
 Corneal ulcers –
 Fusarium,Aspergillus,Alterneria
 Respiratory samples-
 Aspergillus fumigatus ,A.niger,..
 Otomycoses-
 Aspergillus and Rhizopus
 Sinusitis-
 Apergillus fumigatus ,A.niger,..

28. Antifungal resistance
 According to CDC
 Increased R of Candida to
Fluconazole and echinocandins
 Aspergillus R to Azoles
 In our study fluconazole R 37.5%
 Flucytosine-25%
 Amp-B-12.5%
 Echinocandins -8.33%

29. DIAGNOSTIC CHALLENGES & RECENT
ADVANCES
 During the past 3 decades, increase in
population of immunocompromized
patients has increased risk of developing
invasive fungal infections (IFIs)
 Shift in epidemiology
 Increased resistance to antifungal drugs
 Traditional diagnostic methods - cannot
detect until later stages
 Non-invasive diagnostic techniques –
early accurate diagnosis but still need
validation in different patient population

30. Traditional Methods of
Diagnosis
.Direct microscopic examination
-Wet mounts-KOH ,Calcoflour white,India
ink,
Stains-Grams,PAS,Giemsa,GMS,
Fluorescent antibody stain (its use is
limited by the restricted availability of the
specific antisera )

31. Histopathology
- H&E,PAS,GMS
- Mayer’s mucicarmine used to specifically show
the capsular material of Cr. neoformans&
endospores and sporangia of Rhinosporidium
seebri.
- Alcian blue staining - done to demonstrate acid
mucins.
- Advantages- Direct visualization of pathogen
- Infection can be proven if sample
from sterile site
- Disadvantages – Requires invasive techniques
- Specific identification of
pathogen difficult

32. Culture
- Conventional & Automated
- Vitek-2,Maldi-Tof
- - Advantages- accurate, specific
- Considered gold standard
- Wide-scale use in most labs
- Disadvantages :
- May require invasive
techniques to obtain specimen from
sterile site
- May be falsely negative

33. Traditional Methods of
Diagnosis..
3 Non-culture methods • Serology •
Antibody detection • Antigen detection •
Immunohistochemistry.
Serological tests
- Developed & standardized for
Cryptococcus and Histoplasmosis
- Not reliable for other pathogens
-Latex agglutination test for antigen
detection
Tube agglutination tests –

34. NON INVASIVE METHODS
1. High-resolution computed
tomography scans
2. 1–3–β–D -glucan test
3. Galactomannan test
4. Polymerase chain reaction

35. Latest techniques
 Some of the latest techniques
employed in the detection of Fungi,
including FISH-fluorescence in situ
hybridization, DNA array technology,
multiplex tandem PCR, real-time PCR,
PCR-ELISA, RAPD, and
loop-mediated isothermal amplification
(LAMP)

36. High resolution CT scans
 Pulmonary fungal infections
(Aspergillosis) – 100% predictive value
 Prognostic indicator
 Disadvantages – Can’t differentiate
Aspergillosis from other filamentous
fungal infections like mucormycosis

37. SERUM TESTS
 Detect components of fungal cell wall
to diagnose invasive fungal infections.
 Two types
1. (1-3)-beta-D glucan assay
2. Galactomannan test

38. 1-3)-beta-D glucan assay
- Effective pan-fungal marker to aid in screening &
diagnosis of IFIs
- Used for Candida, Aspergillus spp. And Fusarium
spp.
- Not useful for Zygomycetes, Blastomyces,
Cryptococcosis
- high degree of accuracy, sensitivity (97%)
specificity (93%) in AML and myelodysplastic
syndromes.
- Primarily found useful in excluding IFIs due to high
NPV (97.8%) and relatively high proportion of false
positive results (10%)
- Also useful in therapeutic monitoring
- FDA approved kits – Fungitell , useful in leukemic
patients but sensitivity lower in neutropenic
patients
- Standardization and Validation needed for routine

39. Galactomannan test
 - Serological test for Invasive Aspergillosis &
patients with prolonged neutropenia
- According to one study, Sensitivity- 71% and
Specificity- 89%
- False positive :- Feeding with soyabean
- Bifidobacter colonization
- Beta lactam therapy
especially
Piperacillin-tazobactam
- False negative :- Antifungal therapy
(decreases
release of galactomannan
)

40. Tests for detecting cell-mediated
immunity
 Skin testing used for establishing
etiological diagnosis esp. in the
nonendemic area, for epidemiological
surveys and as a prognostic indicator,
as a positive skin test may revert back
to negative with severe or
disseminated diseases. Skin testing is
done in Aspergillus fumigatus, H.
capsulatum, Sporothrix schenkii, and
others

41. Skin tests ..
 It can give false positive reaction in
endemic regions and because of
cross-reactions due to sharing of
antigens. False negative can occur
due to the early stage of infection and
defective CMI.

42. MOLECULAR DIAGNOSTIC METHODS
 Analyses fungal DNA and improve diagnosis
 Increased application but still not accepted as
a diagnostic criterion to define IFIs.
-identifies the genus/species of the pathogen
 RT PCR – can detect genus or species
specific DNA markers like heat shock protein
90
 Pan Fungal PCR tests-for highly conserved
genomic sequences -18S sRNA,28S rRNA or
mitochondrial genes , found in multiple copies
in nearly all fungal species.
 Studies show varying sensitivity (45-
92%),high specificity(>90%).
 Contamination by airborne spores-FPV.
 Does not distinguish colonization from
invasive infection.

43. Commercial molecular assays
for the detection of fungal
pathogens
1) MycAssay™ Pneumocystis (Trinity
Biotech,Ireland)
-Real-time PCR
-Pneumocystis jirovecii mlSU-mitochondrial large
subunit(BAL; sputum)
2) MycAssay™ Aspergillus
-Trinity Biotech Real-time PCR
-18S rRNA; Aspergillus sp.(BAL; serum)

44. CANDIDA
3) Seeplex®ACE PCR system (Seegene Diagnostics,
S.Korea)
-Multiplex PCR coupled with electrophoresis separation
with fluorescence detection(Blood)
4) T2Candida (T2 Biosystems,USA)
-Nanoparticles and T2 magnetic resonance detection
platform
-ITS2(internal transcribed spacer) (Whole blood)
5) Prove-it™ sepsis assay (Mobidiag ,Finland)
-Multiplex PCR coupled with microarray
-ITS; Candida sp. Blood
6) Yeast Traffic Light (AdvanDx ,USA)
-Method=PNA–FISH(peptide nucleic acid fluorescent in-
situ hybridization)
-Target=26S rRNA(Blood -from positive blood culture
bottles)

45. Multiple fungi
7) Luminex xTAG fungal assay (Luminex Molecular
Diagnostics, Canada)
-Multiplex PCR coupled with bead probe fluid array
-Up to 23 fungi(blood and BAL)
8) RenDx™ Fungiplex panel (Renishaw
Diagnostics,UK)
-Multiplex PCR coupled with surface-enhanced
resonance Raman scattering detection
-Up to 50 fungi( Blood)
9) ICEPlex 16-plex fungal panel ( PrimeraDx,USA)
-Multiplex PCR coupled with sequential separation of
amplicons by capillary electrophoresis and multicolor
quantitative detection
-Multiple fungi (Blood)

46. DNA array hybridization
 DNA array hybridization, also known as
reverse dot blot hybridization or macroarray,
is a technique based on hybridization of
amplified and labeled genome regions of
interest to immobilized oligonucleotides
spotted on a solid support platform.
 It is considered a powerful and practical
technique for the detection and identification
of Fungi and other microbes, such as
bacteria, from complex environmental
samples without the need for isolation in
culture.

47. DNA array..
 Synthesized oligonucleotides are spotted
onto a supporting platform, such as a nylon
membrane or glass slide, either manually or
robotically. A positive reaction between an
amplicon and a perfectly matched
oligonucleotide generates a
chemiluminescent signal, which can be
detected by a digital camera in dark rooms.
 With the unlimited capacity for the
accommodation of oligonucleotides on one
membrane and the reusability of the
membranes, it shows superior multiplexing
detection capability at a lower cost over other
PCR-based methods.

48. Loop mediated isothermal
amplification
 Loop-mediated isothermal amplification is a
powerful and novel nucleic acid amplification
method that amplifies a few copies of target
DNA with high specificity, efficiency, and
rapidity under isothermal conditions (do not
require a thermal cycler), using a set of four
specially designed primers and a DNA
polymerase with strand displacement activity.
LAMP assays have been developed for the
rapid detection of pathogenic Fungi like
Penicillium marneffei, Fonsecaea agents of
chromoblastomycosis, Cryptococcus spp.,
Pseudallescheria, and Scedosporium species

49. RAPD
 Random amplified polymorhic DNA
markers are DNA fragments from PCR
amplification of random segments of
genomic DNA with single primer of
arbritary nucleotide sequence
 Disadvantages-technique is lab
dependent which may affect
reprodicibility

50. Strategy to Accelerate Molecular Test
Standardization & Implementation
3 Models
1. European Aspergillus PCR Initiative model -
standardize the molecular tests through forming a
working group and developing a consensus standard
protocol that everyone can follow
2. Centralization model - getting access to a publicly
available molecular test performed under a Clinical
Laboratory Improvement Amendment (CLIA)-certified
reference laboratory; eg, Varicor–IBT Laboratories.
3. Commercialization model - by using commercial
kits that provide standardized methods and quality-
controlled reagents.

51. Three phases of Strategy
 Phase 1-assay development outcome
-developed by industry partner based on clinical
need or research laboratory.
 Phase 2-clinical validation
-consortium /working group
-conducts multicenter evaluation studies,clinical
trials, generate adequate data for FDA
submission
 Phase 3-clinical outcome
-decision depends on clinical outcome ,
prognosis , therapeutic effect and health care
cost

52. Enhancing fungal detection by
Molecular Tests
Areas of interest:-
 Diagnosis of Fungal Bloodstream
Infections
 Detection of Fungi in Formalin – fixed
and paraffin-embedded tissues (FFPE)
 Detection of Antifungal Drug Resistance
directly from clinical samples.
New advances:- to develop
 Quantitative Molecular Assays
 RNA Detection platforms

53. Detection of Antifungal Drug
Reistance
 Emergence of antifungal drug-
resistant organisms is a growing
concern.
 Incorporation of molecular markers for
testing antifungal drug resistance
directly into clinical samples.
 Appropriate antifungals can be
initiated without delay.

54. Conclusion & Future
Perspective
Conventional microbiological and histological
techniques remain the cornerstone of diagnosis for
fungal infections. However, they may be insensitive
and may also require several days for identification
of particular pathogens. Thus, they have a limited
impact on clinical decision-making, and there is an
urgent need for new and efficient diagnostic
methods, which should be fast, highly sensitive,
and very precise.
With the advance of molecular tools, new fungal
species and new mechanisms of resistance will be
clarified, but these methods will require extensive
validation in clinical studies

55. Thank