Mycology

Mycology
MUDr. Vanda Chrenková
photos: Dana Michalská, Dis.
Jiří Tůma
MVDr. Oto Melter, Ph.D.
ÚLM FN Motol a 2. LF UK
Main topics
•
•
•
•
•
•
•
Diagnostics
Candida spp.
Cryptococcus spp.
Pneumocystis jiroveci
Aspergillus spp.
Mucorales
Dermatophytes
Fungi
–
Yeasts
•
–
Moulds
•
–
Candida, Malassezia, Trichosporon, Saccharomyces,
Cryptococcus
Hyaline, Demaciacieae, Zygomycota, Basiomycota ...
Dimorphic fungi
•
Histoplasma, Blastomyces, Coccidioides,
Paracoccidioides
Fungal cell wall
PREANALYTIC
Specimen collection:
•
Aseptically, sterile container
•
Liquids and tissues rather than swab !
Antibodies detection
•
Aspergillus
–
•
Candida
–
•
aspergilloma, allergic forms
detection of invasive infection
Histoplasma, Coccidioides
Antigen
•
•
•
•
•
Aspergillus - galactomannan – cell wall antigen for
detection of invasive aspergillosis (serum, BAL)
1-3-Beta-D-glucan – panfungal cell wall antigen
(detection of yeasts, P.jiroveci and moulds except of
Cryptococcus and Mucorales)
Cryptococcus –glukoronylxylomannan (CSF, serum)
Candida - mannan – detection of circulating antigen
mannan in invasive candidiasis detection (serum).
Histoplasma, Blastomyces, Penicillium marneffei
Microscopy
•
•
•
detection of spores, pseudomycelium,
mycelium
native preparation, Calcofluor white
(fluorescence), Grocott
histopathology
Microscopy
Aspergillus fumigatus in sputum, Calcofluor White, 200x
Culture
•
•
•
Sabouraud agar
37/30/25°C
48 – 72 hours (10 days - 6 weeks moulds)
PCR
•
•
DNA extraction - mechanical and enzymatic disruption of
fungal cell wall
panfungal PCR
Identification
•
•
•
•
•
mass spectrometry
Germ tube test
chromogenic agars
biochemical identification
microscopy of moulds
GERM – TUBE test
quick identification Candida albicans.
•
instructions:
–
–
–
in tube with 0,5 ml horse serum put the isolate (1-3 colonies)
incubation 2-2,5 hours at 37°C
in native microscopy search for germ tubes
•
short filaments
GERM – TUBE test
C. albicans, germ tubes, 400x
Chromogenic agar
• differentiation of Candida albicans and Candida nonalbicans, mixed cultures detection, based on
colorimetric differentiation of colonies in 24 – 48
hours
Candida albicans
Candida krusei
Biochemical identification
AuxaColor™ 2 (Bio-Rad):
•
33 species of yeasts
•
15 tests, 13 assimilation of saccharides and alcools
Antifungal susceptibility testing
(AFST)
•
•
•
in high risk of patient's harm by non-effective
therapy
Disc diffusion test
MIC (microdilution or E-test)
Imprinting microscopy preparation
Microsporum canis, isolate,
200x
Rhizopus sp., isolate,
200x
Candida sp.
•
•
world-wide most frequent cause of fungal infection
yeasts are in nature on organic substrate and
colonising plants, animals and humans
• antibiotics lead to inhibition of normal microflora
and increase in frequency of colonisation and
infection especially in hospitalised patient
• forming blastospores, pseudomycelium and
mycelium
Taxonomie
•
Candida sp.: Bennett 1844, published Berkhout, C.M., 1923
Taxonomic classification
•
Kingdom: Fungi
•
Phylum: Ascomycota
•
Subphylum: Saccharomycotina
•
Class: Saccharomycetes
•
Order: Saccharomycetales
•
Family: Debaryomycetaceae
•
Genus: Candida
Candida sp.
VIRULENCE Factors
• growth at 37°C and thermal dimorfisms
• production of urease, protease, katalase, elastase, fosfolipase
• Th-2 response stimulation
• polysaccharide capsule
• adhesion to organic and anorganic surface
• switch spores to hyfy – infiltration of epithelial cell surface
• cell wall – hydrophobic a resistant to immunity
• phenotypic switch – quick adaptation to environment
changes
Occurence
•
•
ubiquitous (continual exposition): leaves, water, soil,
excreta
Human: 25 – 50% of population – healthy carriers
–
–
skin: C. parapsilosis, C. tropicalis, M. furfur
mucosa of gastrointestinal and urogenital tract: C.
albicans, C. tropicalis, C. glabrata
Sources of Candida
• endogenous
– normal flora
– disruption of natural barriers, multiplication in GIT
and damage of intestinal mucosa
• exogenous
– hands of personnel
– contamination of fluids and supplies
– foreign tissues – valves, cornea
Risk factors
• demographic:
– age (neonates, elderly)
– gravidity
• local factors:
– oedema, maceration, humidity, microtraumatism,
previous colonisation
• other risk
– neutropenia, diabetes, malnutrition, HIV/AIDS
• therapy
– immunosuppressive
– prolonged ATB therapy > 7 days
Risk factors
• iatrogenic factors
– intravenous catheters, drains,
– total parenteral nutrition
– artificial ventilation
– dialysis
– organ transplantation
– big surgery – abdominal, cardiosurgery
– hospitalisation (ICU)
Pathogenesis
•
•
•
colonisation
local infection
disseminated infection
Forms of infection/disease
•
•
•
Mucocutaneous
Systemic and organ
Candidaemia
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanopostitis
Oropharyngeal candidiasis
•
•
•
•
•
•
Asymptomatic colonisation: 25-50% healthy
population
↑ children, elderly
HIV CD4+ < 200/mm3
Aggressive chemotherapy
Corticosteroids
Broad spectrum antibiotics
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanopostitis
Oesophageal candidiasis
•
•
•
•
•
↑ID, HIV CD4+ < 100/mm3
C. albicans
1/3 association with oropharyngeal form
Diagnosis endoscopic: erytema, white
pseudomembranes, non-invasive
Biopsy
Oesophageal candidiasis
•
Complications
–
–
rare
in neutropenic patients
•
•
•
Candidaemia
disseminated candidiasis
Therapy
–
systemic therapy 14 – 21 days
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanopostitis
Anal candidiasis
•
•
•
Itching
In nursling affection of all cheeks = genitogluteal
form in neonates and nurslings
in adults suspicious of ID
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanopostitis
VULVOVAGINAL CANDIDIASIS
•
•
•
•
•
20-50% healthy carriers
2nd cause of vulvovaginal disease
Risk factors: gravidity, diabetes, ATB, AIDS
CD4<200...
↑ C. albicans
Estrogen dependent – ↑ avidity of epithelial
cells to yeasts adhesion
VULVOVAGINAL CANDIDIASIS
•
Diagnostic:
–
–
–
colposcopic: erythema and excoriation; swab of dorsal
vagina wall (10% KOH test, normal pH 4 - 4,5)
microscopy: epithelia, leucocytes, lactobacillus, yeasts
culture
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanoposthitis
CANDIDA BALANITIS A
BALANOPOSTHITIS
•
•
•
•
•
Mucosal erythema with white-grey
pseudomembranes, itching
Affection of glans penis and prepuce
Risk factors: uncircumcised, obese, fimosis, diabetes,
malhygiene, infection of sex. partner
Complication: edema, fimosis
Therapy: local/systemic azoles
Mucocutaneous forms
•
•
•
•
•
•
•
oropharyngeal
oesophageal
anal
genital
cutaneous
vulvovaginal
balanitis a balanopostitis
Intertrigo
•
•
Erythema with white coating
Localisation:
–
–
•
intertriginous
interdigital
Specimen collection:
–
–
scratching or swab from boarding of lesion
culture, event. microscopy
Onychomycosis
•
•
•
Candida ↑ hands (dermatofytes ↑ foots)
Candida onychomycosis usually starts after
paronychial affection, proximally, expansion on
whole nail, possible onycholysis
Specimen collection
–
•
from transition of lesion into healthy parts, by scalpel
Diagnostic
–
–
microscopy
culture
System and organ forms
•
•
•
•
•
•
Hepatosplenic candidiasis
C. of ren and uropoietic tract
C. of respiratory tract
C. endocarditis
C. peritonitis
C. endophthalmitis
UROPOETIC
•
Risk factors:
–
•
Epidemiology:
–
–
•
↑age, female, gravidity, DM, obstructive uropathy,
cancer, instrumental procedures – catheterisation,
colonisation GIT, ATB, immunosupresion, intravenous
catheters
1,3% of candiduria is associated with candidaemia
10% candidaemia origins from Candida uroinfection
Aetiology:
–
C. albicans 50%, C. glabrata 25%, C. tropicalis
UROPOETIC
•
Diagnostic:
–
•
quantitative culture of urine
Therapy:
–
removal of permanent urinary catheter
spontaneous resolution
–
fluconazole
–
Kandidóza dýchacích cest
•
•
•
•
z dutiny ústní
obvykle u hospitalizovaných
u ventilovaných až 40% asymptomaticky
kolonizovaných
infekce DCD velmi vzácná – hematogenní diseminací
Candida endocarditis
•
on damaged valves – defect or surgery – Ao, Mi
–
until 2 month after surgery
→ symptoms – mycotic embolisation
Candida sepsis
• Infection of central venous catheter (CVC)
• Candidaemia
Rex, J. H., T. J. Walsh, and E. A. Anaissie. 1998.
Fungal infections in iatrogenically compromised hosts.
Adv. Intern. Med. 43:321-371.
Infection of central venous catheter
• ↑ affinity to artificial surfaces
• 50 % of positive blood cultures originates
from long-term intravenous catheter and the
infection is transient
• the removal of the catheter is strongly
recommended
Candidaemia
• 50 % transient – CVC related
• Mortality 30 - 40 %, if therapy started later than 12 hours
since the blood culture positivity
(Ostrosky-Zeichner 2011)
Candidaemia
• Diagnostic
– blood culture – „gold standard“
• sensitivity cca 50 %
– antigen mannan – sensitivity 50 %, specificity 60 %
– antibodies
• combination of antigen and antibodies -↑ sensitivity
and specificity
– PCR
Candidaemia
• Therapy
– international guidelines
– national guidelines
– Empirical:
• echinocandin
• fluconazole – de-escalation
Cryptococcus spp.
General
Cryptococcus:
• San Felice 1895, Vuillemin 1901
• saprophytic aerobic yeast round, 3-8 um
• mucopolysaccharid capsule, no mycelium
Epidemiology
• Cryptococcus neoformans (→2 species)
– opportunistic, worldwide distribution
– origin: pigeon droppings, bat guano and the soil
contaminated
• C. gattii (→5 species)
– worldwide, decaying wood >30 trees
– C. gattii a C. deuterogattii - primary pathogenic –
infections in immunocompetent patients
– other species opportunistic
Pathofysiology
• spores inhalation → pulmonary infection (also
unapparent) → hematogenic spread to CNS and
menings
• ↑ neurotropismus – diffuse or bounded lesions
(meningoencephalitis, cryptococcoma)
• rarely skin, bone, joints
CRYPTOCOCCOSIS
Forms
• pulmonary – can be asymptomatic
– acute – in HIV/AIDS
– chronic
• disseminated – from lungs
– CNS – meningitis - neurotropisms
– skin
CRYPTOCOCCOSIS
Diagnostic
• microscopy – Parker ink (Indian Ink) – budding
encapsulated yeasts in CSF (4-6 µm)
CRYPTOCOCCOSIS
Diagnostic
• culture – 30°C, Sabouroud 2-5 days (3 months) –
mucous yellowish big colonies
CRYPTOCOCCOSIS
Diagnostic
•
serology
– antigen – very good sensitivity
– antibodies – low specificity and sensitivity
Therapy:
• pulmonary form in immunocompetent
–
•
self-curable
meningoencefalitis
–
–
amfotericin B + flucytosine
in stable state fluconazole
Pneumocystis jiroveci
• worldwide opportunistic microorganism
• formerly epidemics in neonatal units - malnutrition
(1950)
• HIV
• immunodeficiency primary and secondary
• interhuman transfer
• pathofysiology: thickening of interalveolar septa,
hypoxemia, respiratory insufficiency
Pneumocystis jiroveci
• clinically
– dry cough, fever, progressive dyspnea
• X-ray : diffuse bilateral infiltrates
• Dg. BAL
– microscopy
– PCR
• Therapy
– trimetoprim 20mg/kg/den + sulphametoxazol
100mg/kg/day in 3-4 doses 3 weeks
• Prophylaxis
– co-trimoxazol 800 mg 3x a week
Moulds
•
•
•
Heterogenic group
Thick cell wall, chitin and cellulose
Primary pathogenic – exotic moulds endemic
– Blastomyces, Histoplasma, Coccidioides,
Paracoccidiodides
• Opportunistic pathogens
– Aspergillus, Fusarium, Mucor....
• Dermatophytes
– Epidermophyton, Trichosporon, Microsporum
Most frequent opportunistic moulds
• Aspergillus
– A. fumigatus, A. flavus, A. niger, A. terreus
• Jiné hyalinní houby
– Fusarium, Acremonium, Scedosporium,
Paecilomyces
• Mukormycety
– Rhizopus, Mucor, Rhizomucor, Lichtheimia,
Cunninghamella
• Dematiaceae
– Alternaria, Cladophialophora, Exophiala
Risk factors
• local:
– oedema, maceration, humidity, microtrauma,
• systemic
– oncological or haematological disease
– neutropenia
– diabetes
– COPD, asthma
– lung transplant
Risk factors
• iatrogenic
– immunosuppressive therapy
– artificial ventilation
– organ transplant
– abdominal or cardio surgery
– burns
– ICU stay
Aspergillus sp.
•
•
•
•
•
Ubiquitous, frequent contaminant
2nd most frequent cause of IFD
Aerial transmission, contact
2-9% of hospitalised patient are colonised
Forms
– pulmonary
•
•
•
invasive aspergillosis (acute/ chronic necrotising)
allergic pulmonary aspergillosis (asthma, allergic alveollitis, ABPA)
aspergilloma
– other
• rhinocerebral aspergillosis
• disseminated aspergillosis (gut, liver, kidney, endocard, skin...)
Aspergillus sp.
• Diagnosis
– Microscopy –
• septate hyphae, dichotomic branching, sporulation in presence of
the air
– Culture – undemanding, mycelium pigmented
– antigens – galactomannan, beta-D-glucan
– PCR
• Therapy: voriconazole
Mucormycetes
• Family Mucorales
• Sporadic infections, invasive
–
–
–
–
local – skin and soft tissue
pulmonary
other - sinuses, GUT, CNS...
disseminated
• Angioinvasivity, very quick growth and progression,
multiresistance to antifungals
Mucormycetes
• Diagnostic
– Microscopy – large non-septate hyphae, right-angle
branching, ribbon-like
– Culture – undemanding, at 37°C
– PCR
• Therapy (Chamilos 2008)
– Amphotericin B
– surgery
Prophylaxis of opportunistic mycoses
• Patients in preliminary prolonged neutropenia
• hepaphiltres a overpressure rooms
• elimination of dust from houses
• elimination of plants (living or cut)
• respirators
• monitoring of environment
• antifungals
Dermatophytes
• affinity to keratin (epidermis, nails, hair) →
superficial infections
• Trichophyton, Epidermophyton, Microsporum
Source
• Antropophilic – interhuman contact, contaminated
floor (pool, shower...)
• Zoophiles – pets, livestock
• Geophilic – wound in contact with soil
• swimming pool, sea water, beach sand
Clinic
• Skin
– circular lesions
– Intertrigo
• Hair – tenia
• Sycosis barbae – folliculitis
• Nails
– distal/lateral onychomycosis
Diagnostic
• Specimen collection
– 15 days after any treatment (hair), 2 months after
treatment (nail)
– from active lesion
• Microbiology
– Microscopy – mycelium
– Culture – very slow (1 - 3 weeks)
Systemic antifungals
• polyens
– amphotericin B
• azoles
–
–
–
–
fluconazole
voriconazole
posaconazole
isavuconazole
• echinocandins
– caspofungin, micafungin, anidulafungin
Thank you for your attention