CANDIDA/ YEAST/GERM TUBE TEST

1. Candida
Dr Neelam Gupta
M.D Microbiology
Associate Professor

2. Headings covered
 Introduction -Yeast
 Morphology of Candida
 Species of Candida
 Virulence Factors of C. albicans
 Predisposing factors
 Infections caused by candida albicans
 Candida auris (C. auris)
 Candidiasis – Laboratory diagnosis
 Candidiasis- Treatment
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3. Introduction -Yeast
 Yeasts are single-celled budding
organisms. They do not produce
mycelia.
 colonies are usually visible on the plates
in 24-48 h.
 Their soft, moist colonies resemble
bacterial cultures rather than molds.
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4. Candida albicans
Ultrastructure
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5. 
CANDIDIASIS (Candida albicans)

 There are many species of the genus Candida that cause
disease.
 The infections caused by all species of Candida are called
candidiasis.
 Candida albicans is an endogenous organism.
 It can be found in 40-80% of normal human beings. It is
present in the mouth, gut, and vagina. It may be present as
a commensal or a pathogenic organism.
 Infections with Candida usually occur when a patient has
some alteration in cellularimmunity, normal flora or
normal physiology.
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6. Morphology
 Candida is unicellular yeast fungus.
• It is imperfect, reproducing by budding
• Morphology
 Microscopy: Budding yeast cells, and Pseudohyphae.
 Culture: Creamy colony, fast growing on Sabouraud
Dextrose agar (SDA), Blood agar (48 hr)
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7. Species
 There are many species of Candida
(>150)
 The common species are:
Candida albicans,
C.parapsilosis
C.tropicalis,
C.glabrata,
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9. Virulence Factors of C. albicans
 Adherence
 Dimorphism
 Germ tubes
 Rapid switching of expressed phenotype
 Surface hydrophobicity
 Interference with phagocytosis, immune defences and
complement
 Extracellular hydrolases (Proteinases, lipases)
 Synergism with certain bacteria
 Killer toxins
 Acidic metabolites
 Growth rate and undemanding nutrient requirement
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10. Predisposing factors
 Patients with decreased cellular immunity
have decreased resistance to fungal infections.
 Prolonged antibiotic or steroid therapy
destroys the balance of normal flora in the
intestine allowing the endogenous Candida to
overcome the host.
 Invasive procedures, such as cardiac surgery
and indwelling catheters, produce alterations in
host physiology and some of these patients
develop Candida infections
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11. ‣ Although it most frequently infects the skin and
mucosae ,Candida can cause pneumonia,
septicemia or endocarditis in the immuno-
compromised patient.
‣ The establishment of infection with Candida
species appears to be a property of the host - not
the organism.
‣ The more debilitated the host,the more invasive
the disease .
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12. Candidiasis
 Definition:
 Any infection caused by any species of the yeast
fungus Candida.
 The most common invasive fungal infections in
immunocompromised patients
 4th most common cause of nosocomial blood
stream infection
 It is considered opportunistic infection
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13. Transmission of Opportunistic Fungi
 ENDOGENOUS
 Colonization precedes infection
 Antibiotic suppression of normal flora, fungal
overgrowth
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15. Infections caused by candida albicans
 Mucous membrane infections
 Thrush (oropharyngeal)
 Esophagitis
 Vaginitis
 Cutaneous infections
 Paronychia (skin around nail bed)
 Onychomycosis (nails)
 Diaper rash
 Chronic mucotaneous candidiasis
children with T-cell abnormality
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16.  Oropharyngeal Candidiasis
 Oral thrush:
 White or grey Pseudomembranous patches on
oral surfaces especially tongue with underlying
erythema.
 Common in neonates, infants, elderly
 In immunocompromised host, e.g. AIDS.
 Esophagitis
 Vulvovaginitis :
 Common in pregnancy, diabetics, use of
contraceptives.
 Thick discharge, itching, irritation . Lesion appear
as white patches on vaginal mucosa.
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17.  Intertriginous candidiasis:
 Infections of skin folds eg. axilla, buttock, toe
web, under breast.
 Erythematous lesion, dry or moist or whitish
accompanied by itching and burning.
 Nail infections:
 Onychomycosis and paronychia
 Diaper rash
 Chronic mucocutaneous candidiasis
Cutaneous infections
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18. Oral thrush
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19. Other infections by Candida
 Urinary tract infection
 Candidemia
 Disseminated (systemic, invasive) infection
 Endophthalmitis (eye)
 Liver and spleen
 Kidneys
 Skin
 Brain
 Lungs
 Bone
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20.  Primary pneumonia is less common and could be a
result of Aspiration
 Secondary pneumonia commonly seen with
hematogenous candisiasis
 Immunocompromised patients
 Isolation of Candida from sputum, BAL is not always
significant
 Clinical features
 Radiology,
 Other Lab investigations
Pulmonary Candidiasis
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21.  Increased colonization (endogenous or exogenous
factors)
 Damage in host barriers by catheters, trauma,
surgery
 Immunosuppression
 Central venous catheters (CVC)
 Disseminated candidiasis (involvement of any organ)
 Septic shock
 Meningitis
 Ocular involvement (retinitis)
 Fever could be the only clinical manifestation
Candidemia
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23. Candida auris (C. auris) is a type of yeast
that can cause severe illness and spreads
easily among patients in healthcare
facilities.
It is often resistant to antifungal
treatments, which means that the
medications that are designed to kill the
fungus and stop infections do not work.
Candida auris (C. auris)
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24. Symptoms
C. auris can cause infections in different parts of
the body such as in the bloodstream, open wounds,
and ears.
The symptoms depend on the location and
severity.
Symptoms may be similar to symptoms of an
infection caused by bacteria.
 ‘Colonization’
Someone who is colonized can still
transmit C. auris onto surfaces or objects
that they contact, which can then spread
it to other patients.
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25. Risk Factors
C. auris mostly affects patients with severe
underlying medical conditions and requiring complex
medical care like invasive medical devices like
breathing tubes, feeding tubes, catheters in a vein,
or urinary catheters.
Healthy people without these risk factors, including
healthcare workers and family members, have a low
risk for getting infected with C. auris
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26. Why is C. auris concerning?
• It can cause serious infections.
• It is often resistant to medicines, making it
difficult to treat.
• It is becoming more common.
• It is difficult to identify by routine lab tests. • It
can spread in hospitals and nursing homes.
Resistance and Treatment
C. auris is often resistant to commonly used
antifungal medications, most C. auris infections
are treatable with a class of antifungal medicines
called echinocandins
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27. Specimen depend on site of infection.
Swabs, Urine, Blood, Respiratory specimens, CSF,
Blood
1. Direct microscopy :
Gram stain, KOH, Giemsa, GMS, or PAS stained
smears.
Budding yeast cells and pseudohyphae will be seen in
stained smear or KOH.
Candidiasis – Laboratory diagnosis
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28. 2. Culture:
Media: SDA & Blood agar at 37oC,
Creamy moist colonies in 24 - 48 hours.
3. Blood culture
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29. Because C. albicans is the most common species to cause
infection
The following tests are used to identify C. albicans:
1.Germ tube test : Formation of germ tube when cultured
in
serum at 37ᵒC
2. Chlamydospore production in corn meal Agar
3. Resistance to 500 μg/ml Cycloheximide
 If these 3 are positive this yeast is C.albicans,
If negative, then it could be any other yeast,
 Use Carbohydrate assimilations and fermentation.
Commercial kits available for this like: API 20C, API 32C
 Culture on Chromogenic Media (CHROMagar™ Candida)
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30.  Candida albicans
Sabouraud Agar
Morphology: Creamy white yeast,
may be dull, dry irregular and
heaped up, glabrous and tough
 Chromagar
producing green pigmented colonies
on specially designed medium to
speciate certain yeasts based on
color they produce
Candida species
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31. Germ tube test
 Germ tube is a young hypha (short, non-nucleated
and nonseptate) growing out of a yeast cell or
spore (in sporereleasing fungi) during their
germination.
 They are ½ the width and 3 to 4 times the length of
the cell from which they arise.
 It is a rapid method for identifying and differentiating
albicans from other Candida spp.
 Buds and pseudo-hyphae show constrictions at the
point of origin from cells, unlike germ tubes.
 Germ tube formation was first reported by
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32.  Principle of Germ Tube Test
 When cells of Candida are incubated in protein
rich medium like human, sheep or fetal bovine
serum at 370C for 2-4 hours,
 Candida albicans produce short, slender, tube like
structures called germ tubes.
 Germ tubes can be detected with a wet mount
KOH
 Increased synthesis of protein and ribonucleic
acid results in the formation of germ tubes.
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33.  Procedure: Put few drops of serum (human,
sheep or fetal bovine) or media into a test tube.
 Take a sterile wooden applicator or loop and touch
a colony or two of yeast and gently emulsify it in
the serum.
 Incubate the tube for 2-3 hours at 37°C. Using a
Pasteur pipette, place a drop of the suspension on
a slide and cover with a coverslip.
 Examine the wet mount microscopically (at 40X)
for production of germ tubes
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35.  Result and interpretation:
 Positive Result: A short hyphal (filamentous)
extension arising laterally from a yeast cell with no
constriction at the point of origin, no nucleus and
the tube being half the width and 3 to 4 times the
length of the yeast cell.
 Examples: Candida albicans and Candida
dubliniensis.
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36.  Negative Result: No hyphal extension
arising from a yeast cell or a short hyphal
extension with the constriction at the point
of origin.
 Examples: Candida tropicalis, Candida
glabrata and other yeasts
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37.  Limitations: Candida tropicalis may produce hyphal
extension with constriction at the point of origin after 3
hrs of incubation i.e. pseudo germ tubes, which may
be falsely interpreted as germ tubes.
 Too heavy inoculum will inhibit the formation of germ
tubes.
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38. Yeast Identification
Carbohydrates assimilation test , API 20C
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39. 4. Serology:
Patient serum
Test for Antigen ,
e.g. Mannan antigen using ELISA
Test for Antibodies
5. PCR
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40.  Oropharyngeal:
 Topical Nystatin suspension, Clotrimazole troches ,Miconazole, Fluconazole
suspension.
 Vaginitis:
 Miconazole, Clotrimazole, Fluconazole
 Systemic treatment of Candidiasis
 Fluconazole
 Voriconazole
 Caspofungin
 Amphotericin
In candidemia :
Treat for 14 days after last negative culture and resolution of signs and
symptoms
Remove catheters, if possible
Candidiasis- Treatment
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41. Points to consider:
C. glabrata can be less susceptible
or resistant to fluconazole
C. krusei is resistant to fluconazole
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42. Thank you
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