Opportunistic Mycosis

Presented by Sisay Fesseha, MSc, Diagnostic Biomedicine

Outline of presentation

 Candidiasis
 Asperigilosis
 Zygomycosis
 Cryptococosis
 Pneumocystis carni
Introduction

 The opportunistic mycoses are;

 caused by fungi that cannot infect healthy humans but can
cause serious often fatal mycoses in people whose resistance
has been lowered (immunocompromised patients).
 The highly susceptible groups for opportunistic fungal
infection are
 AIDs patients,
Leukemic patients,
individuals on chemotherapy for treatment of cancer,
alcoholics.
Introduction cont’d…

 Manydiabetic peoples are also susceptible to

pulmonary infection by common saprophytic
mold of the genera Rhizopus and Mucor (both
Zygomycetes) and Aspergillus respectively.
 Unless the predisposing conditions are
corrected, many opportunistic mycosis can be
fatal, even with antibiotic treatment.
Introduction cont’d…

 Many fungi previously considered non- pathogenic are

now recognized as etiological agents of the
opportunistic fungal infections.
 The laboratory must identify and report completely
the presence of all fungi recovered from
immunocompromised patient, since every organism is
a potential pathogen.
Introduction cont’d…
 Although there is an ongoing list of opportunistic mycosis,
They are often seen in three forms that includes :
1. Yeast forms;
Candidiasis,
Cryptococcosis,
2. Mold forms;
Aspargillosis,
Zygomycosis
Mucormycosis
3. Pneumocystis carinii
1. Candidiasis

Candidiasis is a relatively common human infection that can

take form of;
 superficial,
 mucocutanous or
 systemic disease.
 Principally it is caused by the three species of genus candida,
namely,
 C.albicans,
 C.tropicalis and
 C.krusei
Cont’d……

 Candida albicans is the most common cause of

infection.
 Since the species of candida, including C.albicans are
the part of the normal endogenous microbial flora,
candidiasis is usually the result of autoinfection during
a metabolic or an immunologic disturbance.
 C.albicans is not a dimorphic fungus, can exist in the
form of yeast & hyphae in tissue (in vivo).
Pathogenesis and clinical pictures

A. Superficial and mucocutaneous candidiasis

 It is superficial infections of skin and mucous membranes
 Through, oral and vaginal candidiasis
 Oesophagial candidiasis
 Skin lesions of folds, groin, axilla, and interdigital areas
 Napkin eruptions in infants
 Paranychial candidiaiasis
Oral candidiasis
Clinical forms

 Invasive:
 Candidemia: initial stage can be transient if phagocytic
system is intact.
 Disseminated or hematogenous candidiasis if phagocytic
system is compromised.
 Multi organs can be involved with infection: kidney,
prosthetic heart valves, brain, eye, meninges.
 Mortality: 30-40%
Predisposing factors

 Diabetes
 Immunosupperession
 T-cell immunodeficiency disorders
 Acquired- immunodeficiency syndrome, (AIDS)
 Leukaemias, Lymphomas
 Steroid treatments
 Broad spectrum antibiotics
Laboratory diagnosis

 Superficial or mucocutaneous candidiasis is diagnosed by

finding the fungus in tissue scraping and culture

 Systemic candidiasis is difficult to diagnose.

 Definitive diagnosis is made by the histopathologic

demonstration of the invasion of tissue by the yeast.

 Specimens from surface lesions, mouth, vaginal, sputum,

exudates etc are examined using different methods
Direct examination

a) KOH
 Exposed lesions can usually be easily diagnosed by
clinical appearance together with finding typical budding
yeast cells and pseudohyphae and /or true hyphea in lesion
scrapings treated with KOH.

b) Gram- stain
 Gram stain smears shows large gram positive budding yeast cells
with pseudohyphea.
c) Germ tube test

 Candida albicans can be presumptively identified based

on the production of a germ tube
Principle
 When incubated with serum at 370C for 1 to 3 hours,
C.albicans will form a germ tube.
Procedure
1.Pipette 0.5 ml of serum into a test tube
2. Inoculate the tube with a small amount of the
organism to be
tested.
Germ tube test cont’d…

A large inoculums may produce false- negative results.

3. Incubate at 370C for 1 to 3 hours
4. Place a drop of the suspension on a slide, apply a cover
slip, and examine for the presence of germ tubes.
 Interpretation
 A germ tube is approximately half as wide and three to
four times as long as the yeast cells.
Germ tube test

Germ tube negative

Germ tube positive
Germ tube test cont’d…

 No point of constriction should exist where the germ

tube arises from the mother cell.

 C.stellotidae is also germ tube positive.

Quality control
 C. albicans: positive control; produces germ tube
within 2 hours

 C.tropicalis: negative control; fails to produce germ

tube within 3 hours
d) Culture and identification

 Growth is rapid on sabouroud’s dextrose blood agar, triptase

soy, and many other media.
Creamy yeast colonies are formed after overnight incubation at
temperature of 210C or 370C; the optimum growth
temperature is around 300C.
 C.albicans grows as round-to-oval yeast cells that are 4-6µm
in diameter.
 Pseudohyphae and hyphae also seen; especially at lower
incubation temperature (i.e. 220C-250C) and on nutritionally
poor media.
C.albicans on SDA culture media

The different species are

further identified on the
bases of their biochemical
properties.

 Note: Asexual reproduction

is by budding of yeasts or
by chalmydospore
formation on hyphae. C.albicans on SDA culture media
2. Aspergillosis
Aspergillosis

 Aspergillosiscomprises a group of world wide

human disease caused by about dozens of fungal
species of the genus Aspergillus. About 185
different species has been identified.
 Since Aspergillus species are many and are
widespread in soil, air, and decaying vegetable
matter many people are heavily exposed to
inhalation of spores.
Aspergillosis
 Pathogenesis and clinical picture

 The spectrum of human disease caused by Asperagillus ranges

from allergy to disseminated infection.
 Most of the disease processes are caused by the inhalation of
conidia.
 Allergy:- Persons may develop IgE. against Aspergillus and
present with allergic condition. Example, asthma
 Non-invasive colonization:- Aspergillus can colonize a pre-
existing lung cavity e.g., tuberculosis, forming fungus ball or
aspergilloma.
Invasive aspergillosis

 May occur in severely

immunocompromised patients, organ transplant
recipients,.

 Patients under corticosteroid therapy and patients

with leukaemia.

 Toxin production
 e.g. aflatoxin of Aspergillus flavus is
carcinogenic; cause liver cancer and necrosis. Can be
found in spoiled legumes.
Predisposing factors

 Infections occur principally in individuals who have

disease or predisposing abnormalities such as cystic
fibrosis, bronchietasis, chronic bronchitis, tuberculosis
etc. or who are immunosuppressed.

 Aspergillus species are not dimorphic; they are

characterized by chains of conidia borne or condiophore.

 Speciation is based on principally morphology; some 150

species are recognized.
Predisposing factors

 Infections occur principally in individuals who have

disease or predisposing abnormalities such as cystic
fibrosis, bronchietasis, chronic bronchitis, tuberculosis
etc. or who are immunosuppressed.

 Aspergillus species are not dimorphic; they are

characterized by chains of conidia or condiophore.

 Speciation is based on principally morphology; some 150

species are recognized.
Predisposing factor cont’d…

 Aspergillus fumigatus is most commonly

recovered from immunocompromised patients,
and most often seen in clinical laboratories.
 Aspergillus niger and A. flavus are also
recognized as significant cause of infection in
immunocompromised patients.
 Laboratory diagnosis

 The diagnosis of aspergillosis is difficult due to their wide

spread in nature.
 Direct examination
 Microscopicexamination of KOH preparations of sputum
show characteristic branching hyphae filaments and may
show the conididophores attached to the spare head.
 Demonstration of hyphal fragments in tissue biopsy is
diagnostic of tissue invasion
Gram stain of Aspergillus
Aspergillus in tissue showing acute angle branching, septate hyphae
Culture and identification

 A.fumigatus is rapidly growing mold (2 to 5

days) that produce a fluty to granular, white to
blue- green colony, on blood enriched medium.
 Maturesporulating colonies most often exhibits
the blue- green powdery appearance.
Aspergillosis examined under a microscope
Aspergillosis in a culture media
Serologic test

 Antibodies to Aspergillus extracts are

detected in serum and positive skin
testing in allergic cases.
3. Zygomycosis (phycomycosis,
mucormycosis)

 Zygomycosis is caused by several genera belonging

to zygomycetes, e.g. mucor, rhizopus, and Abiedia.
 It is some what less common infection when
compared with Aspergillosis; however it is a
significant cause of morbidity and mortality in
immunocompromised patients.
Cont’d ………

 LikeAspergillus species, members of

the zygomycetes are common
environmental molds of low intrinsic
pathogencity.
 The disease occurs in debilitated persons, metabolic acidosis,
malignancies, uraemia, malnutrition, steroid therapy and
hepatic failure.
 The organisms produce broad non septate hyphea, aerial
mycelia, sporangia, sporangiospores and sexual zygospores;
grow rapidly on routine media to produce profuse cottony
colonies.
 Pathogenesis and clinical picture

 One of the most common forms observed in the

 Rhinocerebral form in which the nasal mucosa, plate,
Sinuses, orbit, face, and brain are involved; each shows
massive necrosis with vascular invasion and infraction.
 Other types of infection involved the lungs and
gastrointestinal tract; some patients develop
disseminated infection.
Cont’d …….

 Organs including the liver, spleen, pancreas,

and kidney may be involved.
 The hyphae usually invade the upper or lower
respiratory tract or through burn wound of the
skin.
 Lesion progression is usually rapid, spore do not
form in vivo.
Laboratory diagnosis

 Isbased on the demonstration of non-

septate hyphae in smears and biopsy
sections.
 Culture show sporangium containing
sporangiospores
Morphology of frequently isolated opportunistic
fungi

Zygomycetes
Aspergillus species
Rhizopus sp. showing sporangium and rhizoids.
4. Cryptococcosis

Definition :
 The causative organism is cryptoceccus
neoformas Cryptococcosis occurs in sporadic
form and as an opportunistic infection
associated with immunosupperession.
 The organism is found in soil contaminated with
bird excreta particularly pigeons.
 Infection occurs by inhalation that results in
lung infection (Cryptococcus pneumonia).
 The infection spreads to the central nervous
system and meninges (cryptococcus meningitis).
 Transmission of cryptococcosis

Natural history of saprobic and parasitic cycle of Cryptococcus neoformans.

Laboratory Diagnosis

 Detection of Cryptococcus neoformans in sputum and CSF.

 The organism is characterized by the presence of large

gelationous carbohydrate capsule that surrounds the budding
yeast cell.

 Direct examination
 The capsulated yeast cells are best seen in an India ink
preparation (wet mount)

 Immunofluorescent stain is applied to dried smear.

C. neoformans gram stain and indian ink
stain

Gram stain of C. neoformans Indian ink stain C.neoformans

Laboratory diagnosis cont’d…

 Culture and identification

 The organism grows easily giving rise to
mucoid colonies with the typical capsulated
yeast cells.
The organism is ureases positive.
 Serologic test
Capsular antigens are detected in C.S.F and in
the circulation by using anticapsular
antibodies.
Cryptococcus neoformans India ink preparation demonstrating the large capsule surrounding budding yeast cells
5. Pnemocystis carinii
 The taxonomy of P.carinii remains in question, with
evidence of classification as both a fungus and a
protozoan.
 Ribonuclic acid (RNA) sequencing, cell wall composition,
ulterstructure, and method of reproduction seem to
indicate the organism is fungus.
 P.carinii may thus be reclassified with in the fungi
species. P.carinii is wide spread I nature and often found
in several mammals; especially rodents, cats, dogs,
sheep, and goats.
Forms of Pneumocystis carinii

The organism has two forms

 Cyst stage
 It measures 5-8µm in diameter
 May contain up to eight oval intera-cystic bodies
 Trophic or pleomorphic form
 It measures 1-4µm in diameter
 Covered with tiny projections for attachment to the
host epithelial cells.
 The cyst form is inhaled and the intra-cystic bodies
released in to the alveoli of the lungs.
 The tropic form develops and multiplies
asexually on the surface of the epithial
cells.

 The trophic form than rounds up forming a

cyst stages. None of the stage is intera
cellular
Pathogenesis and clinical picture

 P.carinii produces a symptomatic pneumonia in

debilitated or premature infants who are
immunodeficent.
 A rapid, progressive pneumonia is also seen is
patients with leukemia, following transplants, and
most recently in AIDS patients.
 In fact. P.carinii pneumonia was one of the first
opportunistic pathogens linked to AIDS.
Pathogenesis and clinical picture

 To days, more than half of all AIDS

patients have P.carinii infection and it
is remains a leading cause of death in
these patients.
 The infection may disseminate in AIDS
patients in to the eyes, liver, spleen or
bone marrow.
Laboratory Diagnosis
 Silver stain of P. carinii
Direct microscopic
examination
 Microscopic observation of
the cyst or trophic form in
lung secretion or lung tissue
after staining using Giemsa stain showing Pneumocystis carinii
toluidine blue O, Modified
Gomori methenamine silver
nitrate, or Giemsa.
Treatment of Opportunistic fungal infections

 Oral thrush & mucocutaneous

 Topical nystatin
 Oral ketoconazole
 Fluconazole
 Systemic
 Amphotericin B
 Oral fluconazole
 Prevention: remove moisture, drugs, personal hygiene, avoid birds excreta ,
wear masks
Summary

 Write the predisposing factors for candidiasis?

 List the laboratory test used to diagnose candidiasis?
 How do you differentiate candida albican from other
candida species?
 Explain the clinical presentation of Aspergillosis?
 What is the major route of infection of Cryptococcosis?
Any Question?

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