Opportunistic mycoses, histoplasmosis, sporothrix schenkii, Dimorphic fungi lab diagnosis,

1. Systemic & Opportunistic Mycoses
Dr Mohammed Ashraf Ali
MD (Micro)
KIMS HUBLI

2. Terminologies
• Dimorphic fungi
• Systemic fungal disease
• New world

3. SYSTEMIC MYCOSES

4. Definition
Deep seated fungal infections caused by fungi that are soil
saprophytes.
• Infection is accidental.
• Inhalation of air borne spores produced by moulds
• Dimorphic fungi are causative agents.
• Ex:
– Blastomyces dermatitidis
– Paracoccidioides brasiliensis
– Coccidioides immitis
– Histoplasma capsulatum

5. Blastomycosis
(North American blastomycosis)
Chronic infection, characterized by
• Formation of suppurative and granulomatous lesions in any
part of the body
• With a marked predilection for the lungs and skin
Causative agent: Blastomyces dermatitidis
Distribution:
1. North America
2. Africa
3. India- Delhi (bronchial aspirates of patient and lungs of
insectivorous bats)

6. Mode of Transmission & Epidemiology
• Source of infection- soil
It grows in moist soil rich in organic material, forming hyphae
with small pear-shaped conidia
• Mode of infection- Inhalation of the conidia
• Primary pulmonary pathogen & resembles TB or histoplasmosis
• M:F ratio- 4:1
• 20-50 yrs age group
• Occupation- farmers and tree cutters

7. Clinical forms
1. Pulmonary form- Gilchrist disease, Chicago disease
– Asymptomatic
– Productive cough, weight loss, chest pain and fever
– Chronicity- resembles carcinoma, TB
– Dissemination
2. Cutaneous form
– Traumatic inoculation into the exposed parts
– Papule/ nodule breaks down to form a fistula- discharge pus
3. Disseminated form
– In immunocompromised individuals
– Other sites affected- bones and genitourinary organs

8. Laboratory diagnosis
A. Specimen-
sputum, pus, biopsy
transport asap….
B. Processing of specimen
1. Microscopy
2. Culture
3. Skin test
4. Ab Detection
5. Nucleic acid Detection- DNA probes
6. Animal pathogenicity – Mice, rats used to study virulence

9. 1. Microscopy- KOH (10%) or Calcofluorwhite
Yeast form:
• Large yeast cells
(8-12 μm)
• Blastoconidia
attached by broad base
• Double contoured wall
Mold phase:
• Lollipop forms

10. 2. Culture
Media : SDA, BHI agar, Blood Agar
• Two media inoculated
• incubated- one at 25°C and one at 37°C for 4 weeks.
Colony on medium at 25°C- Fluffy and tan coloured
• LPCB - septate hyphae with oval or pyriform conidia (2-10µ)
Chlamydospores are thick walled
Colony on medium at 37°C- Cream coloured, smooth,
• LPCB- thick walled yeast like cells, with broad based budding
Confirmation of the isolate
• Conversion of mycelial form to yeast form on BA at 37°C
• Exoantigen analysis

11. 3. Skin testing
• DTH to blastomycin,
• not of much value in diagnosis

12. 4. Antibody Detection
• Complement fixation test
• Precipitation-Immunodiffusion
• ELISA
• RIA

13. Prevention & Control
• Amphotericin B & Ketoconazole- for Rx
• Surgical excision
• There are no means of prevention

14. Paracoccidioidomycosis
• South American Blastomycosis or Brazilian blastomycosis
Definition: Chronic infection, characterized by
• Formation of suppurative lesions in any part of the body
• With a marked predilection for the lungs and skin
Causative agent: P. braziliensis
Distribution:
1. South America, esp. Brazil
2. India- not yet reported

16. Mode of Transmission & Epidemiology
• Source of infection- soil
It grows in moist soil rich in organic material, forming hyphae
with small pear-shaped conidia
• Mode of infection- Inhalation of the conidia, no man to man
transmission
• Primary pulmonary pathogen & resembles respiratory disease
• M:F ratio- females less affected due to estrogen
• 20-50 yrs age group

17. Clinical forms
1. Pulmonary form- Gilchrist disease, Chicago disease
– Asymptomatic
– Dissemination is by hematogenous route
2. Mucocutaneous form
– Cooler areas of body such as nasal and oropharyngeal
– Ulcerative lesions seen in mouth, on lips, tongue and conjunctiva
3. Lymphatic Paracoccidioidomycosis
– Cervical lymphadenopathy and can spread to other LN
4. Disseminated
– Seen in Immunocompromised patients
– Disease spreads to other organs specially adrenals

18. Laboratory diagnosis
A. Specimen-
sputum, pus, biopsy, bronchoalveolar lavage
transport asap….
B. Processing of specimen
1. Microscopy
2. Culture
3. Skin test- paracoccidioidin
4. Ab Detection- CFT, ELISA, counter immunoelectrophoresis
5. Nucleic acid Detection- DNA probes, PCR
6. Animal pathogenicity – Mice, rats used to study virulence

19. 1. Microscopy- KOH (10%) or Calcofluorwhite
Yeast form:
• Yeast forms with multiple buds
encircling mother cell.
• Mariner’s wheel or pilot’ wheel or
mickey mouse cap appearance
• 15-30µ
• Narrow based budding

20. Mold phase: Lollipop forms

21. 2. Culture
Media : SDA, BHI agar, Blood Agar
• Two media inoculated and incubated- one at 25°C and one at 37°C.
• Slow growth- 6 weeks
Colony on medium at 25°C- Fluffy and tan coloured
• LPCB- septate hyphae which are sterile (no conidia)
Colony on medium at 37°C- Cream coloured, smooth,
• LPCB- mariner’s wheel
Confirmation of the isolate
– Conversion of mycelial form to yeast form on BA at 37°C
– Exoantigen analysis

22. Treatment
• Itraconazole, fluconazole with amphotericin B and
ketoconazole

23. Coccidioidomycosis
• Causative agent- Coccidioides immitis
• Dimorphic fungus
• Primary pulmonary pathogen
Distribution
• Endemic in southwest USA and northern Mexico

24. Source of infection- Inhalation of arthrospores

25. Clinical features
• Majority – asymptomatic
• Pulmonary disease which resembles mild
influenza like fever (Valley fever) to
Pneumonia
• Dissemination- uncommon but highly fatal

26. Laboratory diagnosis
Specimens
• Sputum
• Pus
• Biopsy

27. Direct examination- Microscopy
Yeast form
• Appears as a spherule(15-75µm in diameter)
• Thick double walled refractile wall filled with
endospores.
• Each endospore- spherule.
Mycelial form
• Pseudohyphae which fragments into arthrospores-
highly infectious.

28. Culture
• SDA or BHI agar with cycloheximide and
chloramphenicol
• Incubate at 37˚C and 25 ˚C
• Warning
arthrospores are highly infectious- arthrospores are
borne, never use petridishes for culture

29. Skin test
• DTH (similar to tuberculin)
• Ag- coccidioidin
• Interpretation
positive test (5mm induration within 48 hrs)
• Endemic areas test not useful

30. HISTOPLASMOSIS
• Also k/a Reticuloendothelial cytomycosis/ Caver’s
disease/ Spelunker’s disease/ Darling’s disease
• Causative agent- H. capsulatum
• Dimorphic fungus
• Disease of Reticuloendothelial system
• Intracellular parasite
• Distribution- Worldwide, most common in America

31. Source of infection
• Soil enriched with excreta of birds or bats
• Inhalation of spores
Reticuloendothelial system.. How???
• Lymphadenopathy
• Hepatospleenomegaly
• Fever and anemia

32. Clinical features
• Majority – asymptomatic
• Some- pulmonary disease- resembles tuberculosis
• Dissemination
Any skin or mucosal lesions??
• Granulomatous and/ or
• Ulcerative lesions

33. Laboratory diagnosis
Specimens
• Sputum
• Bone marrow aspirates
• Peripheral blood
• Skin scrapings
• Lymph node of biopsies and biopsy of other
organs

34. Direct examination- Microscopy
• Staining: Giemsa or Wright stains
• H.capsulatum appears as small oval yeast
cells, (2-4µm in diameter) packed within the
cytoplasm of macrophages or monocytes.

35. Culture
• SDA or BHI agar with cycloheximide and
chloramphenicol
• Incubate at 37˚C and 25 ˚C
• Yeast forms- 37 ˚C
• Mycelial forms- large thick walled spherical spores
with tubercles or finger like projections at 25 ˚C

36. Serological tests
After two weeks of infection- antibodies detected
• Latex agglutination test
• Precipitation test
• Complement fixation test
Rise in the antibody titre- progressive disease

37. Histoplasmin skin test
• DTH (similar to tuberculin)
• Ag- Histoplasmin
– Culture filtrate of Mycelial phase of H.capsulatum
• Interpretation
positive test ---- indicates past/ present infection
Treatment-
Amphotericin-B

38. African Histoplasmosis
• H. duboisii
• Africa
• Primarily involves skin and subcutaneous tissues
• Lungs not involved.
• Morphologically similar to Mycelial phase of
H.capsulatum
• Larger and elongated yeast forms

39. Opportunistic Fungal
Infections

40. Opportunistic Mycosis
• Opportunistic mycosis is a fungal or fungus-like disease
occurring in a human with a compromised immune system.
• Opportunistic organisms are normal resident flora that become
pathogenic only when the host's immune defences are altered,
– immunosuppressive therapy,
– in a chronic disease, such as diabetes mellitus,
– steroid or antibacterial therapy that upsets the balance of
bacterial flora in the body.

41. Common Opportunistic Fungus
• We find the highest frequency of opportunistic
fungal infections come in the following order:
1.Candidiasis
2.Aspergillosis
3.Cryptococcosis

42. Candida as Opportunistic
Infection

43. Candidosis
• Candidiasis / Monoliasis,
• Normal flora
Exist in Mouth, Gastrointestinal
tract, Vagina, skin in 20 % of
normal Individuals.
• Called as Yeast Like fungus
• Currently important cause of
opportunistic fungal infection.

44. Morphology and Culturing
• Ovoid shape or spherical budding cells and
produces pseudo mycelium
• Routine cultures are done on Sabouraud's
Glucose agar,
• Grow predominantly in yeast phase
• A mixture of yeast cells and pseudo mycelium
and true mycelium are seen in Vivo and
Nutritionally poor media.

45. Macroscopic and Microscopic
appearance of Candida spp

46. Pseudohypal structures in Candida

47. Systemic Candidosis
• Occurs in Patients who carry more yeasts in Mouth, GIT
• Predisposed with Individuals with
1. On antibiotic or/and Steroid Therapy
2. Immunosuppressed
3. Recipients with organ transplantation
4. Infancy – Old age – Pregnancy
5. Indisposed with trauma Occluding lesions,
6. Diabetes mellitus.
7. Zinc and iron deficiencies

48. Predisposition after Surgery and
Therapeutic Approaches
• Post operative Immuno
Suppression
• Use of IV catheters
• Use of cytotoxic drugs
and cortosteriods
• Use of Urinary
Catheters

49. Important species of Candida in
Human infections
• C.albicans
• C.tropicalis
• C.glabrata
• C.Krusei

50. Prominent Infections with
Candida

51. Oral Thrush produced by
Candia albicans

52. Many cases of AIDS are suspected
by observation of Oral Cavity

53. Laboratory Diagnosis
• Skin scrapings,
• Mucosal scrapping,
• Vaginal secretions
• Culturing Blood and other body fluids,
• Observations
Microscopic observation after Gram staining.
Gram + yeast cells.

54. Laboratory Diagnosis
• Isolation of Candida
from various specimens
confers diagnosis
• Serology
• Molecular Methods

55. Microscopy
• Gram staining – A rapid
method
• KoH preparation
• Methylamine silver
staining

56. Culturing
• Easier to culture on
Sabouraud's dextrose agar
• Culturing in routine
Blood culture Media
• Culturing urine - A semi
quantitative estimations
are essential Colony
forming units essential in
attributing infections

57. Easier Identification of
species as C.albicans
• Germ tube test
identifies C.albicans
from other Candida
species.
• Majority of Diagnostic
laboratories depend on
this test.

58. Emerging Methods for detection of
Candida Infections
• Molecular Methods
• PCR

59. Cryptococcosis.

60. Cryptococcus neoformans
• A Capsulated yeast – A
true yeast..
• A sporadic disease in
the past.
• Most common infection
in AIDS patients.

61. Structure of C.neoformans

62. Morphology
• A true yeast
• Round 4 – 10 microns
• Capsule - Mucopolysaccharide.
– Negative staining with India Ink and Nigrosin
• KOH preparations in Sputum and other tissues,
• PAS and Mucicaramine staining helps confirmation.

63. As Seen in India Ink preparation

64. Life cycle of C.neoformans

65. Laboratory Diagnosis.
• Sample- CSF
• Direct microscopy – IIP, Gram staining
• Cultures on SDA,
• Serological tests for detection of Capsular antigen
– Latex test for detection of Antigen
– Blood cultures
– ELISA

66. Treatment
• Immune competent - Fluconazole, Itraconazole
• Immune Deficient – Amphotericin B
Flucytosine

67. ASPERGILLOSIS

68. Aspergillosis
• In nature > 100 species of Aspergillosis exist, Few are
important as human pathogens
1 A.fumigatus
2 A.niger
3 A.flavus
4 A.terreus
5 A.nidulans

69. Fungal spores enters through
respiratory tract

70. Pathogenesis clinical presentations
• Allergic Aspergillosis – Atopic individuals, with elevated IgE
levels
• 10-20% of Asthmatics react to A.fumigatus
• Allergic alveoitis follows particularly heavy and repeated
exposure to larger number of spores
• Maltsters Lung – causes allergic alveolitis, who handle barley
on which A.claveus has sporulated during malting process

71. Pathogenesis
• Aspergilloma – A fungal
ball, fungus colonize
Preexisting cavities in the
lung and form compact ball
of Mycelium which is later
surrounded by dense fibrous
wall
• C/F- cough with
expectoration, Haemoptysis

72. Culture
• Cultured as Mycelial fungus
• Separate hyphae with
distinctive sporing structures
• Spore bearing hyphae
• Sterigmata

73. Zygomycosis

74. Zygomycosis
• Saprophytic mould fungi
• Major Causative agents
– Rhizopus,
– Mucor,
– Absidia.

75. Mucor
• Microscopy
Non septate hyphae
Having branched
sporangiophores
with sporangium at
terminal ends
• No rhizoids

76. Rhizopus
• Microscopy
Shows non septate
hyphae
Sporangiophores in
groups
they are above the
Rhizoids

77. Important Clinical
Manifestations
• Rhino cerebral Zygomycosis
associate with Diabetes
mellitus, leukemia, or
lymphomas
• Causes extensive Cellulitis,
and tissue destruction.

78. Mucormycosis
• Cellulitis causes
extensive tissue
destruction.
• Spread from Nasal
mucosa to turbinate
bone, paranasal sinuses ,
orbit, and Brain
• Rapdily fatal if
untreated

79. Other Manifestations
• Severe immuno compromised may manifest as
primary cutaneous lesions
• Rarely infects Burns patients
• But lesions can be less severe than Brain
lesions

80. Microscopy
• In KOH preparation shows
broad aseptate branching
mycelium, and distorted
hyphae
• Methenamine silver is more
sensitive.
• Staining with PAS not helpful

81. Culturing
Culture on Sabouraud's dextrose agar.