41.Diagnostic Mycology
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Transcript of 41.Diagnostic Mycology
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PH 152 Diagnostic Mycology
March 13, 2013 Prof. T. de Guzman
Liana, PL, Jo, Laine, RJ 1 of 5
LAST 152 LECTURE FOR THE YEAR WOOHOOOOOO!
Medyo mahaba but thats just because maraming pictures. And sorry for the blocks of text, yun talaga yung nilagay ni maam
sa presentation niya. Dont be discouraged! Kaya natin ito! Push natin yan!
-PL & Liana
[note: most of the pics in this trans were the same ones maam used in her ppt]
Advantages in Diagnostic Mycology
Factors influencing emergence and reemergence of fungal infections
1. Use of broad-spectrum anitbiotics 2. Immunosuppressive therapy 3. Heroin addiction 4. Severe underlying causes
*Global warming due to climate change may influence the behaviour of microorganisms. This is why theres emerging and re-emerging orginisms INTRODUCTION
The correct identification of fungi is of great importance in: o Clinical setting o plant pathology
there are now emerging fungal plants pathogens that are becoming human pathogens
usually saprophytes o biodeterioration o biotechnology o environmental studies
New cases of Fungi affecting humans and animals; even plants (2011)
Myceliophthora thermophila is a thermophilic mold commonly found in:
silage (in drainage)
wood chips
soil
There are rare reports of infection (that mimic aspergillosis)
o Case of mimicking invasive aspergillosis was diagnosed in an immunocompromised ptx in France
Cryptococcus gattii some regard this as a variant of C.
neoformans o but is a separate species as of now o but maam said as of now considered siya as a
separate species)
Human nail pathogens in the genus Arachnomyces
Arachnomyces kanei, (arachno= spider) was described for isolates obtained form abnormal nails of patients
Plant pathogens causing human infection
Macrophomina phaseolina, a plant pathogen
Reported for the first time as the cause of disseminated infection in a Canadian man, originally from Sri Lanka,
who received a renal transplant in India
Patient subsequently succumbed to invasive infection with a different but related fungus, Neoscytalidium dimidiatum (formerly Scytalidium dimidiatum)
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Liana, PL, Jo, Laine, RJ 5 of 5
Molecular data confirmed the identification of both fungi o Revealed that they are related species within the
ascomycete family Botryosphaeriaceae
(How did he become infected? The instruments were probably contaminated)
Mycoleptodiscus indicus, a fungus found in tropical or sub-
tropical regions
Causing septic arthritis of the knee in an otherwise healthy Canadian male o septic meaning haematogenous dissemination
Fungal infection was diagnosed in Canada but thought to have been contracted while the patient was inCosta Rica o probably through direct inoculation through injured
skin o The fungus was isolated from synovial fluid and
identification was confirmed by DNA sequencing. A soil fungus causing fungemia in an immunocompromised host
Graphium basitruncatum, a fungus known previously from
soil in Solomon Island and Japan
Was reported as the cause of fungemia in an immunocompromised patient from Canada.
The fungus was isolated repeatedly from blood cultures and it spread to the skin resulting in necrotic skin ulcer
LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS (note: sabi ni maam, ah wag na to but she went on reading and discussing this slide anyway, so your call)
Clinical suspicion from physical or clinical manifestation of the patient
History of travel
Immune status- is he immunocompromised?
Selection of appropriate specimens
Cultures
Direct microscopic examination
Invasive diagnostic procedures i.e. biopsy, aspirates Serologic tests for antigen, antibody or metabolites
Antifungal susceptibility testing- hard in the Philippines
Interpretation of laboratory results
Communication with the physician
Communication with the laboratory
Morphology
Classification systems of organisms are historically based on observable characteristics o the phenotypic approach o phenotype: expressed characteristic/ morphological
classification
Classification and identification of fungi, relies mainly on morphological criteria both in culture and in microscopy (unlike other important pathogens such as bacteria or viruses)
Cultural Morphology/Characteristics
A. fumigatus
Aspergillus sp. Culture (which you may mistake as penicillum)
Histoplasma capsulatum culture
LIGHT MICROSCOPE
classical LM methods have been enhanced by: o Nomarski differential interference contrast
phase/ contrast microscopy cells are LIVING
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Liana, PL, Jo, Laine, RJ 5 of 5
o Fluorescence o Cytochemistry/histochemistry
Cyto: cells of the fungi Histo: demonstration of the fungus in the tissue
through histopathological techniques o Development of new staining techniques
Alejandro Posadas first described C. immitis in Argentina Domingo Ezcurra first case to manifest coccidioidomycosis; his head is kept in a jar in a museum
C. immitis
common in San Joaquin Valley (California) but can be present in the arid regions of the US o When going to the savannas, you may inhale this C.
immitis Brown males are more susceptible
Candida sp.
note the pseudohyphae
Cryptococus neoformans stained to show the THICK, BIG,
POLYSACCHARIDE capsule
India-ink preparation is used primarily to examine CSF for the presence of the encapsulated yeasts o Ex. Cryptoccocus neoformans
This is an india ink preparation from an exudates containing encapsulated budding yeasts.
ELECTRON MICROSCOPE - Transmission electron microscopy (TEM)
of cell wall cross sections reveal significant differences between: Ascomycetous and Basidiomycetous yeasts
- Scanning electron microscopy (SEM)
Increased availability has greatly facilitated identification at the species level of some
ascomycetes - Freeze fracturing
has revealed fine details of wall layers of conidia or ascospores
freezing using the nitrogen gas - Fluorescence microscopy
To generate chlamydospores/chlamydoconidia of Candida
you must culture in CORN MEAL AGAR as opposed to the regular fungal agar - Sabarauds
Dextrose Agar/ Potato Dextrose agar
HISTOPATHOLOGY
- presumptive diagnosis - use of special stains - Periodic-acid-Schiff (PAS) - Gomori-methenamine silver (GMS)
Pneumocystis pneumonia caused by Pneumocyst carinii (previously classified as protozoansremember patho!) DIAGNOSTIC PITFALLS
When using GMS stains, normal tissue structures can appear as yeasts or hyphae o Neurosecretory granules o Collagen fibers, with one even showing a
psedoseptum o In speciments w/ few organisms, hyphae cut
transversally can appear as yeasts that may have pseudobudding
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Liana, PL, Jo, Laine, RJ 5 of 5
E TEST
Left: test for sensitivity of Aspergillus to Itraconazole at different concentrations o In this case, the Aspergillus was resistant
Right: test for sensitivity to Voriconazole o Sensitive! (strip with clearing zone around it)
PROTEIN IDENTIFICATION AND QUANTIFICATION
Determination of protein concentration on microtiter plates (changes color like in ELISA)
Application of separated peptides to a sample carrier for Mass spectrometry
PHYSIOLOGICAL & BIOCHEMICAL
Physiological and biochemical techniques are applied, as has routinely for years not for the filamentous
Growth rates on defined media under controlled conditions are also valuable in studies o for complex genera like Penicilium
Commercially available kits such as the API system SEROLOGICAL TARGETS
Serology methods utilize the reactions and properties of serum o Antibodies use of commercially available antigens o Antigens use of specific antibodies
Fungi usually extrude exo-antigens
exo-antigens are secreted out antigens are inherent in their body
Why use serology? Because its faster than culturing the organism! Available serologic tests *remember that fungus are spores-bearing (lalo na C. immitis) so they are part of biosafety level 3 Antibodies
- Immunodiffiusion: antigen-antibody reactions through diffusion
- ImmunoCAP
IgE & IgG can be detected
ABPA (Antigens in Bronchopulmonary Aspirates of): o asthma, cystic fibrosis, COPD, cavitary
disease
Test principle: measurement of specific IgE antibodies w/ ImmunoCAP ISAC
o results in a broad spectrum allergen profile on a molecular level
- Radioallergosorbent Test (RAST): IgE are detected
Antigens - Latex Agglutination
was used a long time ago in detection of Cryptococcus neoformans antigens in CSF and blood
- Radioimmunoassay (RIA) Antibodies and antigens - Complement fixation (remember 151!) - Enzyme-linked immunosorbent assay (ELISA) - Enzyme Immunoassay (EIA) DIAGNOSTIC TOOLS 2011
- Sandwich-ELISA
Galactomannan
Mannan o because the structure of the wall of the fungus
are composed of glucose and mannose which are very antigenic
- High-resolution CT-scan (Computed Tomography) - Ultrasound - Bronchoalveolar lavages
washings of lower and upper respiratory tract - Biopsy techniques - Blood Cultures - Fungitell
kit to detect the B-1-3-D-glucan structure of fungi
- PCR
for identification of the organism through amplification of the gene/DNA encoding for something
gene coding for 16S rRNA usually the one used for identification
Aspergillus precipitin test
[picture: line of identity, line of partial identity, non-identity]
Strong reactions: indiciative of Aspergilloma Exo-antigen Identification System
For the culture of Blastomyces, Coccidioides and Histoplasma by immunodiffusion
Immunocap
IgE, IgG, ABPA (asthma, cystic fibrosis, COPD, cavitary disease o Ex. of cavitary disease Aspergiollosis causing
fungus ball & histoplamosis
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Liana, PL, Jo, Laine, RJ 5 of 5
(1-3)-B-D-glucan
Widely distributed in nature (fungi, yeast, algae, bacteria, and plants
Not present (or low) in: o Cryptococcus species o Zygomycetes
o Humans because we dont have that on our cell wall
Exo-antigenthat can be shed
May also be used in candidiasis or fusariosis G test
For broad spectrum detection of fungal infection
Specific test for (1,3) B-D-glucan present in most fungal
cell walls
Relies on the factor G component of horseshoe crab amoebocyte lysate which is sensitive to glucan
Very sensitive test
(+) result: clotting of the crabs blood (no need to know the principle involved)
Fungitell Assay
Highly sensitive, rapid diagnostic test
Detects (1-3)-B-D-glucan in serum in as little as one hour DIAGNOSIS OF INVASIVE DISEASE
Galactomannan (GM) o Polysaccharide component of cell wall o Highly immunogenic antigen o Present in most Aspergilli o Exo-antigen that can be detected in serum,
Broncheoalveolar Lavage or CSF
Monitoring of GM during antifungal therapy allows progression of teatment to be measured
Several commercially available ELISA tests o Platelia, Pastorex
HOWEVER GM presence in patients blood is determined by multiple factors o Sensitivity of GM detection depends on the site of
infection o Certain antibiotics may give false positive result
ampicillin, amoxicillin, amoxicillin- clavulanate Aspergillosis should also be confirmed by other diagnostic
tools such as CT scan
CANDIDIASIS
Antigen test MANNAN Highly immunogenic anitgen
Immunologically more active than B-glucan
Polysaccharide component of the cell wall of Candida
Positive results may be obtained 2- 15 days before positive blood culture
Commercially available ELISA tests o Like Pastorex and Platelia
HOWEVER!! Negative results of tests do not mean exclude infection
Antibody Test
Sensitivity ~80% in immunocomponent individuals
Anti candida antibodies may also be present in healthy individuals and cause false positive results
CRYPTOCOCCOSIS
Primary aetiological agent: C. neoformans
Diagnosis: only based on detection of capsular polysaccharide antigen (glucuronoxylomannan)
NO antibody tests performed
Several tests:
Latex agglutination o PREMIER Cryptococcal antigen assay
Enzyme immunoassay o Pastorex, Crypto Plus, IMMY Latex Cryptococcus
antigen assay
Marker Method Availability
IgA, IgG, IgM ELISA Commercial kits
Galactomannan ELISA Commercial kits
Glucan Colorimetric Commercial kits
Glucan ELISA Investigational
DNA PCR Investigational
*According to maam these do not hold true anymore, pero diniscuss pa rin niya eh LATERAL-FLOW DEVICE FOR RAPID SERODIAGNOSIS OF INVASIVE ASPERGILLOSIS
- like a pregnancy test - 2 bands: positive
Last trans of our first year na puro majors! Sobrang bilis no? After this medyo makakahinga na tayo then last year in PH! Haha. Thank you for an awesome year, 2014!