Respiratory mycosesRespiratory mycoses(reduced version)(reduced version)

Dr Elizabeth M. Johnson, DirectorDr Elizabeth M. Johnson, Director

HPA Mycology Reference Laboratory, HPA Mycology Reference Laboratory,

BristolBristol

Kingdom FungiKingdom Fungi

Eucaryotic organismsEucaryotic organisms No chlorophyllNo chlorophyll Absorb nourishment from living or dead organic Absorb nourishment from living or dead organic

materialmaterial Filamentous form: septate or aseptate hyphaeFilamentous form: septate or aseptate hyphae Yeasts: unicellular fungiYeasts: unicellular fungi Sexual and asexual formsSexual and asexual forms

Inhalation of fungal sporesInhalation of fungal spores

Spores Spores 8 µm8 µm

SINUSITISSINUSITIS

PULMONARYPULMONARYASPERGILLOSISASPERGILLOSIS

DISEASE SPECTRUMDISEASE SPECTRUM

ALLERGYALLERGY

ASPERGILLOMAASPERGILLOMA

DISEMINATEDDISEMINATEDASPERGILLOSISASPERGILLOSIS

2-4 µm2-4 µm

Schizophyllum communeSchizophyllum commune

Basidiomycete often found on rotting woodBasidiomycete often found on rotting wood

Increasingly recognised as a cause of Increasingly recognised as a cause of sinusitissinusitis

Produces raised fruiting bodies on prolonged Produces raised fruiting bodies on prolonged incubationincubation

Look for clamp connections and spiculesLook for clamp connections and spicules

Presence of risk factorsPresence of risk factors

Clinical signsClinical signs

LaboratoryLaboratory

Microscopy & Culture of sputumMicroscopy & Culture of sputum

and BALand BAL

Serology - detection of antibodies Serology - detection of antibodies and fungal antigens and fungal antigens

Detection of fungal DNA Detection of fungal DNA using PCR using PCR

Diagnosis of respiratory mycosesDiagnosis of respiratory mycoses

Patchy changes seen on chest radiograph of Patchy changes seen on chest radiograph of patient with invasive aspergillosispatient with invasive aspergillosis

Aspergillosis: radiological findingsAspergillosis: radiological findingsin the lungsin the lungs

Halo sign on CT scan - early finding (1-10 days)Halo sign on CT scan - early finding (1-10 days)

Crescent-shaped lesions on chest radiograph Crescent-shaped lesions on chest radiograph

- late sign following neutrophil recovery- late sign following neutrophil recovery

Halo sign Halo sign Air crescent Air crescent

Aspergillus Aspergillus hyphae in a hyphae in a sputum samplesputum sample

Microscopy of a Microscopy of a mucoraceous mouldmucoraceous mould

Absidia corymbiferaAbsidia corymbifera

Fungal serology - AntigensFungal serology - Antigens

Latex agglutination + Latex agglutination + ++++++

ELISA ELISA + + ++++++

Tests for antigenaemiaTests for antigenaemia Asp Cryp Asp Cryp

TARGET

• Cell wall componentsCell wall components::

mannans mannans

galactomannangalactomannan

1,3 beta-D-glucans1,3 beta-D-glucans

• Cytoplasmic antigens Cytoplasmic antigens • MetabolitesMetabolites

Aspergillus ELISAAspergillus ELISARat EBA-2 monoclonal antibodyRat EBA-2 monoclonal antibodydirected against galactomannandirected against galactomannan

sensitivity - 1 ng/mlsensitivity - 1 ng/ml

G - testG - testAmebocyte limulus lysate test Amebocyte limulus lysate test Horse shoe crab coagulation Horse shoe crab coagulation

cascadecascadesensitivity - 1 pg/mlsensitivity - 1 pg/ml

Pitfalls of PCRPitfalls of PCR

Choice of sampleChoice of sampleBlood, Serum or PlasmaBlood, Serum or PlasmaBALBALTissueTissue

Method of DNA extractionMethod of DNA extractionMechanicalMechanicalChemicalChemicalEnzymesEnzymes

Contamination:Contamination:Environmental Environmental AmpliconAmplicon

Absence of standardization of reagents and targetsAbsence of standardization of reagents and targets

Infection with Infection with Scedosporium Scedosporium spp.spp.

Pseudallesheriosis - sexual form (teleomorph) Pseudallesheriosis - sexual form (teleomorph) Pseudallescheria boydiiPseudallescheria boydii

Clinically indistinguishable from invasive Clinically indistinguishable from invasive aspergillosisaspergillosis

Specific features:Specific features:

Infection often follows near-drowning Infection often follows near-drowning accidentsaccidents

Neutropenia is a risk factorNeutropenia is a risk factor

Refractory to treatmentRefractory to treatment

Scedosporium apiospermumScedosporium apiospermum

Scedosporium prolificans

Infection with Infection with Fusarium Fusarium spp.spp.

Clinically very similar to invasive aspergillosisClinically very similar to invasive aspergillosis

Specific features:Specific features:

Cutaneous lesions (70%)Cutaneous lesions (70%)

Blood culture often positive (50-70%)Blood culture often positive (50-70%)

Often water borneOften water borne

Sinus infection may lead to disseminated diseaseSinus infection may lead to disseminated disease

Fusarium solaniFusarium solani

Cryptococcosis in the UK Cryptococcosis in the UK 1980 - 19991980 - 1999

0

10

20

30

40

50

60

1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1997 1998 1999

Non-AIDS AIDS

Year

No. cases

AIDS-defining 24 18 24 illness

Knight et al. 1993 J Infect 27:185-191

Cryptococcus neoformansCryptococcus neoformans

CAN infect healthy, non-immunocompromised hostsCAN infect healthy, non-immunocompromised hosts

Today mainly associated with HIV-infected patientsToday mainly associated with HIV-infected patients

AIDS-defining illnessAIDS-defining illness

HAART has had an impactHAART has had an impact

Mode of infection is inhalation but haematogenous Mode of infection is inhalation but haematogenous dissemination leads to meningitisdissemination leads to meningitis

Diagnosis by direct microscopy or LPA antigen testDiagnosis by direct microscopy or LPA antigen test

Indian ink stain of Indian ink stain of Cryptococcus neoformansCryptococcus neoformans

Pneumocystis carinii Pneumocystis carinii var. var. hominishominis= Pneumocystis jiroveci= Pneumocystis jiroveci

Coccidioides immitisCoccidioides immitis

Cause of coccidioidomycosisCause of coccidioidomycosis

Endemic areas southwestern USA, Endemic areas southwestern USA,

Central and South AmericaCentral and South America

Infection by inhalation of arthroconidiaInfection by inhalation of arthroconidia

Mild transient pulmonary infectionMild transient pulmonary infection

1% develop disseminated disease 1% develop disseminated disease

Coccidioides Coccidioides immitis immitis distributiondistribution

Endemic areas: Endemic areas: southwestern USA southwestern USA Central and South Central and South AmericaAmerica

From: From: Medical MycologyMedical MycologyKwon-Chung and BennettKwon-Chung and Bennett1992 Lea & Febiger1992 Lea & Febiger

Coccidioides immitis life cycleCoccidioides immitis life cycle

From: Medical Mycology Kwon-Chung and Bennett 1992 Lea & FebigerFrom: Medical Mycology Kwon-Chung and Bennett 1992 Lea & Febiger

Direct microscopyDirect microscopy

Endosporulating spherulesEndosporulating spherules

seen on direct microscopyseen on direct microscopy

of pus, sputum or joint of pus, sputum or joint

fluidfluid

Difficult to obtain in cultureDifficult to obtain in culture

CultureCulture

Mycelial colonies after Mycelial colonies after

incubation at 25 - 30ºC incubation at 25 - 30ºC

for 2-7 daysfor 2-7 days

Growth on actidioneGrowth on actidione

Typical arthroconidiaTypical arthroconidia

Exoantigen test to confirmExoantigen test to confirm

Gene-probe also availableGene-probe also available

Laboratory diagnosis of Laboratory diagnosis of coccidioidomycosiscoccidioidomycosis

Serology Serology Immunodiffusion and complement fixationImmunodiffusion and complement fixation

HistoplasmosisHistoplasmosis

Guano-enriched soil sourceGuano-enriched soil source

Tissue form: budding yeastsTissue form: budding yeasts

Reticuloendothelial systemReticuloendothelial system

Chronic cases - calcifications Chronic cases - calcifications

Widespread geographicallyWidespread geographically

- especially Eastern USA, & Africa- especially Eastern USA, & Africa

HistoplasmaHistoplasma var. var. capsulatum capsulatum and var. and var. duboisiiduboisii

HistoplasmosisHistoplasmosis

Direct microscopyDirect microscopy

Small, oval, budding yeast Small, oval, budding yeast

cells seen in Giemsa cells seen in Giemsa

stained smearsstained smears

Cells often seen withinCells often seen within

macrophagesmacrophages

CultureCulture

Mycelial colonies after Mycelial colonies after

incubation at 25 - 30ºC incubation at 25 - 30ºC

for 2-7 daysfor 2-7 days

Conversion to yeast on Conversion to yeast on

blood agar or BHI agar at blood agar or BHI agar at

37ºC, 2-6 weeks37ºC, 2-6 weeks

Laboratory diagnosis of histoplasmosisLaboratory diagnosis of histoplasmosis

Serology Serology

Immunodiffusion and Immunodiffusion and complement fixationcomplement fixation

Sensitive and specificSensitive and specific High positive predictive valueHigh positive predictive value High negative predictive valueHigh negative predictive value Simple, rapid and inexpensiveSimple, rapid and inexpensive

The ideal diagnostic testThe ideal diagnostic test