Lateral flow antigen tests and

diagnosis of import mycoses

Katrien Lagrou, PharmD, PhD

University Hospitals Leuven and KU Leuven, BELGIUM

Co

nve

ntio

na

l Te

sts Histopathology

Direct Microscopy

Culture

Mass Spectrometry (MALDI-TOF)

Susceptibility testing

An

tige

n/A

ntibo

dyl/D

NA

Antigen detection

Galactomannan (GM)

1,3 β-D-glucan (BDG)

Lateral Flow Assays (LFA)

PCR

Which tests can the lab offer to you?

Antibody detection

Co

nve

ntio

na

l Te

sts Histopathology

Direct Microscopy

Culture

Mass Spectrometry (MALDI-TOF)

Susceptibility testing

An

tige

n/A

ntibo

dy/D

NA

Antigen detection

Galactomannan (GM)

1,3 β-D-glucan (BDG)

Lateral Flow Assays (LFA)

PCR

Which tests can the lab offer to you?

Antibody detection

Diagnosis of

cryptococcosis and

aspergillosis

• Cryptococcus neoformans var grubii (serotype A),

affects mainly immunocompromised individuals

• Cryptococcus neoformans var neoformans

(serotype D)

• Cryptococcus gattii (serotype B and C), causes

disease mainly in apparently healthy individuals

The pathogen

• Cryptococcus neoformans species complex

• Cryptococcus gattii species complex

OR

• Antigen: the major capsular polysaccharide glucuronoxylomannan

(shed during infection)

• Detection in serum/CSF

• Latex-agglutination/Lateral flow assay

• “One of the most useful serologic tests in mycology”

Cryptococcus antigen detection

• Correlation between antigen and number of yeast cells

• Highly sensitive and specific

– False positive results possible in serum by rheumatoid factor or in

case of infection with Trichosporon asahii

– False negatives:

• Prozone effect

• Organism load is low or organisms not well encapsulated

Detecting cryptococcal antigen in CSF or serum is rapid,

specific, and virtually diagnostic of meningo-encephalitic

or disseminated cryptococcosis.

Cryptococcal antigen test

LFA for Cryptococcal antigen

• Dipstick format

• Glucuronoxylomannan (GXM) has variable levels of O-

acetylation that contribute to serotype specificity

• CrAg LFA high sensitivity for GXM for the four serotypes as

follows A=B>C=D

• Stable at room temperature, costs about 2 $/strip in low-

income countries

• Can not be used to monitor treatment response, clearance of

the cryptococcal capsule polysaccharide by macrophages is

an independent and slower process than killing of the yeast

by antifungal therapy

• More sensitive than culture and latex agglutination

M. A. Gates-Hollingsworth and T.R. Kozel, CVI, 2013, 20: 634-635

LFA provides prognostic value

High correlation between log10 CFU and log2 LFA titers on day 1

T. Kabanda et al. CID 2014, 58: 113-116.

Samples were collected

as part of 2 prospective

cohorts in Uganda

Pre-emptive treatment of cryptococcosis

Window of a median of 22 days during which cryptococcal antigen can be

detected in serum but patients have not yet developed symptoms and clinical

disease

= asymptomatic cryptococcal antigenemia

= independent predictor of mortality in patients initiating ART

Screening recommendation may need expansion to ≤ 200 cells/µL to maximize detection of

patients in whom fluconazole pre-emptive therapy may be life-saving (or only in

combination with certain symptoms?)

KA Magambo et al, JIAS, 2014, 14: 19040

• Murine MAb JF5: IgG3 immunoglobulin

• Recognizes an extracellular, constitutive, glycoprotein antigen

• Antigen is secreted during active growth of hyphae and is not produced by dead

or quiescent spores

• Displays superior specificity to rat MAb EB-A2 (Bio-Rad Platelia GM-EIA)

• Used to develop a rapid, user-friendly, diagnostic test for detection of IA

Thornton CR. Clin Vaccine Immunol. 2008 Jul;15(7):1095-105

The lateral flow device: a point-of-care test

1 Cryptococcus neoformans 2 Candida albicans3 Fusarium solani4 Rhizopus oryzae5 Aspergillus fumigatus

LFA for the diagnosis of invasive aspergillosis

Monoclonal antibody: Mab JF-5

Antibody source: mouse

Step 1

Step 2

Positive reaction Negative reaction

Capillary flow

Step 3

BA

Control line Test line Control line

BA

Aspergillus lateral flow assay

Sample padCapture zone

Clinical specimen

Test line(mAb)

Control line(anti-mouse IgG)

Sample pad Sample pad

Ag No-Ag

Pretreatment of

serum samples

necessary, not of

BAL samples

Current Fungal Infection Reports (2013) 7: 244-251.

Expert Review of Clinical Immunology (2014)

Localisation of JF5 antigen

Commercialisation of the

Aspergillus LFD, when?

• 286 BAL from 221patients with underlying respiratory diseases

(without hematological malignancy or SOT)

• 14% proven/probable IPA

• 02/2012 – 05/2014, Graz, Austria

Prattes J et al. Am J Resp Crit Care Med 2014; 190: 922-929

Novel tests for diagnosis of IA in patients with

underlying respiratory disease

Combined assay performance for blood screening

in high risk hematology patients

Case-control design: 22 proven/probable IA versus 59 controls

White PL et al, JCM 2013, 51: 1510-1515.

Diagnosis of import mycoses:

histoplasmosis

• Pathogen:

– Histoplasma capsulatum var. capsulatum

Most common endemic mycosis in North America

Also Central and South America, Central and South Africa, Asia, Middle

East, Australia, very rare in Europe

– Histoplasma capsulatum var. duboisii

African histoplasmosis – Central and West Africa

• Thermally dimorphic

• Diagnosis

– Direct examination and histopathology of biopsies, respiratory

samples, blood and bone marrow

– Culture: 2-6 weeks incubation needed, molecular identification

Histoplasma capsulatum var. duboisii: thick-walled

yeast cells with narrow-based buds

Yeast form, narrow based buds

Histoplasma capsulatum

Histoplasma capsulatum

Diagnosis of histoplasmosis

– Antibody detection:

• Negative results may be obtained during the first month, repeat testing

• NPV is not 100%

• Immunodiffusion (M and H band: active histoplasmosis, M alone: early or

chronic histoplasmosis, test performed in ITM Antwerp) and

complement fixation tests (more sensitive but less specific)

– Antigen detection:

• Particularly useful in immunocompromised individuals with disseminated

disease

• Sensitivity is greatest with urine (95-100% in aids patients with

disseminated disease)

• Sensitivity about 75% in acute pulmonary histoplasmosis if samples are

obtained within 2 weeks after exposure

• Cross reactions with blastomycosis, paracoccidioidomycosis,

penicillinosis, less frequent in coccidioidomycosis

– Aspergillus Galactomannan may be positive in histoplasmosis

Coccidioidomycosis

• Pathogens: Coccidioides immitis and Coccidioides posadasii

• Most Southwestern US but also Central and South America

• Diagnosis:

– Direct examination/histopathology

– Culture: fast growing in 3-5 days, white – gray to buff

– Antibody detection: CF and ID

• ID:

– with heated coccidioidin as antigen, detects IgM, present in 75% of

patients within 1 week after the onset of symptoms and 90% within 3

weeks

– unheated: detects IgG antibodies, 2-6 weeks after onset of

symptoms

• CF: IgG antibodies, 4-12 weeks after infections, more sensitive but less

specific than the ID test

– Antigen test: sensitivity highest in urine (about 70% in severe cases),

cross reaction with histoplasmosis, blastomycosis and

paracoccidioidomycosis

Coccidioides immitis

D.Wilmes et al, Case report in preparation

Blastomycosis

• Pathogen: Blastomyces dermatitidis

• North-America, sporadic cases in Central America, South

America, the Middle East, Asia, Poland, Italy, …

• Diagnosis:

– Direct examination and histopathology: unipolar budding on a broad

base with a thick wall, free or in giant cells

– Culture: incubate up to 4 weeks, white-brown cottony colonies

– Antibody detection: CF (lacks specificity) and ID (specific and

diagnostic but negative in 10% of patients with disseminated disease

and 60% of localized infections)

– Antigen detection: highest sensitivity in urine, cross reaction with

histoplasmosis, paracoccidioidomycosis, penicillinosis and less

frequently coccidioidomycosis

Blastomyces dermatitidis

Mycology online

Yeasts: large, with broad-budding

• http://miravistalabs.com/

Diagnosis of endemic mycosis:

general considerations

• Also immunocompetent patients may develop disease

(large inoculum)

• BSL-3 for propagating and manipulating sporulating cultures!

• Reversion to yeast phase is an identification criterion but

molecular techniques often provide faster results

• High level of suspicion, especially if a fungus initially grows as

a yeast (37°C) and as filamentous fungus upon subculture

(25-30°C)

• Diagnosis may be unexpected!

• Man, 61 y

• 16/05/2015: Lung transplantation for end stage COPD

• 12/09/2015: Hospitalization because of CMV reactivation

• 14/09/2015: 1 blood culture bottle with fungal hyphae

• ‘Yeast like cells’ grow the next day

• Subculture on diluted Sabouraud:

Penicillium species - a red pigment diffuses in the agar ….

BUT the patient was never in South-East Asia!

BUT the donor was!

Stayed a few weeks in Myanmar Feb 2015

My conclusion: this is most likely Penicillium marneffei

Recent case UZ Leuven

• Additional positive blood cultures on 25/09/2015, 28/09/2015,

30/09/2015 + molecular identification of the fungus as P. marneffei

Organization:

KU Leuven, Department of Microbiology & Immunology

AND

The Belgian Society for Human and Animal Mycology

In collaboration with:

Prof. dr. P.E. Verweij, UMC St. Radboud, Nijmegen,

The Netherlands

The course aims at laboratory technicians and scientists who

want to develop or expand their practical knowledge of medical

mycology. Also medical microbiologists and physicians are

welcome (a more clinically oriented 9 day mycology course

will be organized in September 2017).

Mycology Course: practical information

• 5-day course: September 5-9, 2016

• Place: at the KU Leuven Campus Gasthuisberg.

• Language of the course: The theoretical sessions will be given

simultaneously in Dutch (Dr. Ignace Surmont) and French (Prof. M.P.

Hayette). The practical sessions are supported by highly trained laboratory

technologists.

• Registration fee for the course is 1000 €. This includes the textbook

«Medically Important Fungi: A Guide to Identification, Fifth Edition» from

D. Larone, slides and lunches.

• An assessment test will be organized about 3 weeks after the course.

• The participant receives a «Certificate of medical mycology».

• Registration before 1 May 2016. Maximum 20 students are allowed (the

course was full in 2014 !)

• Registration: margaretha.charlier@uzleuven.be