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)

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

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

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

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!