Serological tests in mycology by DR.A.SIVARANJINI BALAMURUGAN

1. SEROLOGICAL
TESTS IN
MYCOLOGY
A.SIVARANJINI

2. SYNOPSIS
• INTRODUCTION
• HISTORY
• SEROLOGICAL TESTS
• FUNGAL PATHOGENS
• ADVANTAGES AND DISADVANTAGES
• CONCLUSION

3. INTRODUCTION – Need for Serologic tests
• Invasive fungal infections(IFI) present a great challenge
nowadays, esp. d/t expansion of population with
immunosuppression.
• Key elements in improving outcome of invasive fungal
infections is rapid diagnosis & early initiation of appropriate
antifungal therapy.
• Cultures, though the gold standard of diagnosis, is time
consuming and has low sensitivity d/t low concentration of the
organism in the tissues.
• Hence non-culture methods for fungal diagnosis is opted.
• These include- detection of specific host immune responses to
fungal antigens using immunologic reagents.
- amplification and detection of specific fungal
nucleic acid sequences .
- detection and quantitation of specific fungal
metabolite products.

4. • Serological tests have now gained importance d/t
- rapidity of results
- serve as a prognostic indicator
• Serological methods utilise the reactions and properties of
the serum.
• Determine humoral response of the host (skin test, invitro
lymphocyte stimulation test-CMI)
• Targets for serological tests are
- Antigen
- Antibody
- Metabolites
• Decision of fungal serologic test is based on
- clinical presentation
- exposure history
- risk factor for infection

5. FUNGAL PATHOGENS OF MEDICAL
IMPORTANCE
OPPORTUNISTIC
• Candida species
• Aspergillus species
• Cryptococcus species
• Pneumocystis jiroveci
TRUE PATHOGENS
• Coccidioides immitis
• Paracoccidioides brasiliensis
• Histoplasma capsulatum
• Blastomyces dermatitidis

6. HISTORY
• Serologic tests were first applied to the diagnosis of
mycotic diseases in the early 1900’s.
• 1956 – discovery of Limulus Amoebocyte Lysate
• 1978 – Restrepo and Moncada developed Latex
agglutination test for P.brasiliensis
• 1979- First detection of circulating antigen of Aspergillus
fumigatus in sera of mice and rabbits by Enzyme linked
immunosorbent assay.
• 1980 – Development of commercial fungal identification
systems using fungal antigens and metabolites.

7. SEROLOGICAL TESTS IN USE
• AGGLUTINATION
• IMMUNODIFFUSION (ID)
• COMPLEMENT FIXATION TEST (CFT)
• ENZYME LINKED IMMUNOSORBENT ASSAY(ELISA)
• LATERAL FLOW ASSAY
• COUNTER IMMUNO-ELECTROPHORESIS (CIE)
• RADIO IMMUNOSORBENT ASSAY (RIA)

8. AGGLUTINATION
• LATEX AGGLUTINATION TEST
• Grading of the Latex Agglutination test
Negative- milky suspension with absence of agglutination
1+ - small clumpings against a cloudy background
2+ - small to moderately sized clumps against a slighlty cloudy
background
3+ - moderately to large sized clumps against a clear
background
4+ - large sized clumps against a clear background
• Advantages – rapid
- long shelf life (4-6 months)
• Disadvantages
- false positives (Eliminated by pretreatment of crude
exoantigens with sodium metaperioidate or pretreating serum with 2β
mercaptoethanol)

9. DIAGNOSIS
1.Cryptococcosis
• Capsulated yeasts
• Polysaccharide antigen ((glucuronoxylomannan)
Serum>CSF>Urine
• Detection of the capsular antigen is confirmative of Cryptococcal infection
• Qualitative & semi-quantitative test
• Sample – serum , CSF, bronchoalveolar lavage
• Significant titre ≥1:8
• Sensitivity – 90.9%
• Specificity – 95%
• False positives – Rheumatoid factor, Trichosporon asahii, Stomatococcus
• False negative – Prozone phenomenon
• Prognostic marker
• Marketer – Meridian Bioscience

10. 2.Diagnosis of Invasive Candidiasis
• Detection of Heat labile glycoprotein (HLP)
• Detection of Mannan antigen (Pastorex Candida Antigen)
• Detection of anti –Mannan antibody (Pastorex Candida Antibody)
• Sensitivity
• Specificity
• False positives
• False negatives
3.Diagnosis of Paracoccidioidomycosis
• Paracoccidioides brasiliensis
• Sensitivity – 84%
• Specificity – 81%
• False positives – Aspergillosis , Histoplasmosis.

11. IMMUNO DIFFUSION
• Patient samples are placed in wells in an agar plate surrounded
by a larger well containing purified antigen.
• Antibodies (serum) and antigen will diffuse out of their
respective wells and an antigen-antibody complex will form a
visible precipitation band in between the wells .
• The presence of a precipitation band indicates a positive test.
• SENSITIVITY : 80-90% (lower in patients with early or
localized disease)
• SPECIFICITY : >90%
• Disadvantages – costly
• - difficult to standardize
- long turn around timehow long

12. 1. Aspergillosis
• Diagnosis of Allergic Bronchopulmonary
Aspergillosis(ABPA), Aspergilloma ,
• Detection of IgE antibodies
• False positives – seens in healthy individuals in
endemic areas
• Sample
• Sensitivity
• Specificity

13. 3. Candidiasis
• Detection of Anti- Mannan antibodies
4.Blastomycosis
• Blastomyces dermatitidis
• Sample – serum, CSF
• Qualitative assay
• Detection of precipitating antibodies to purified A antigen
• Specificity ≥90%
• Sensitivity – low in early infection or
localised disease
• Long turnaround time how long

14. 5.Histoplasmosis
• Histoplasma capsulatum
• Antigens
 M protein –abundant in mycelial form
 H protein - indicative of active infection
• Antibodies
 Anti M Ab - develop soon after infection
- lasts upto 3 yrs after resolution
 Anti H Ab - alone /with Anti-M Ab indicates active/recent
infection

15. • Sample – serum
• 2 precipitating bands – M and H
• M band alone – active or past infection
• H and /or M band – active histoplasmosis
• Sensitivity – 80-100%
• Specificity >90%
• Significant titre : 1:32
• False negatives - immunocompromised pt.
- early stage of infection
• Marketers – Meridian Bioscience(USA only)
- H.capsulatum serum & antigen

16. 6.Coccidioidomycosis
• Coccidioides immitis /C.posadasii
• IgM – develop to tube precipitin(TP) Ag
- detectable from 3wks – 6 mths of symptoms
• IgG – develop to complement fixation (CF) Ag
- indicates current or past infection
• Detection of IgM & IgG separately
• Sample – serum
• Sensitivity
• Specificity
• Marketers – Meridian Bioscience
- C.immitis F Ag & anti C.immitis F serum
- C.immitis TP Ag & anti C.immitis TP serum

17. COMPLEMENT FIXATION TEST
The ability of antigen-antibody complex to ‘fix’ complement is
made use of in CFT
Antibody(patient sample) + fungal antigen (added)
Antigen –Antibody complex
Inactivates exogenously added complement
specific Ab + specific Ab –
Complement fixed Complement lyse RBCs
RBC pellet

19. 1. Aspergillosis
• Detection of IgE antibodies
• Diagnosis of allergic aspergillosis
• Sample
• Sensitivity
• Specificity
• False positives
• False negatives
• Marketers

20. 2. Histoplasmosis
• Ab detected against M & H antigens
• Determination of endpoint titre
≥1:32 or serially increasing titres – active infection
1:8 -1:16 – presumptive evidence of infection
< 1:8 - insignificant
Declining titres – resolution of infection.
• High sensitivity but low specificity compared with ID
• False positive with low titres- in endemic individuals
• Long turnaround time
• Labour intensive

21. 3.Coccidioidomycosis
• Detects IgG Ab to Coccidioides culture filtrate
• Endpoint titre
≥ 1:16 – severe / disseminated infection
1:8 – 1:16 – acute infection
1:2 – 1:8 – past infection / acute focal infection
• Sample
• Sensitivity
• Specificity
• False positives
• False negatives
• Marketers

22. ENZYME LINKED IMMUNO SORBENT ASSAY

23. 1.Cryptococcosis
• Sample
• Sensitivity
• Specificity
2.Aspergillosis
• Allergic aspergillosis
- detection of IgE Ab
• Invasive aspergilllosis
- GALACTOMANNAN(GM) assay

24. Galactomannan assay
• Polysaccharide released by growing Aspergillus hyphae.
• Screening test for diagnosis of Invasive Aspergillosis
• Sample – serum (neutropenics)
- bronchoalveolar lavage ( neutropenics &
- CSF non neutropenics)
• One stage immuno enzymatic sandwich microplate assay
• Uses rat EBA-2 monoclonal antibodies, directed against
circulating Aspergillus Galactomannan(exo antigen)
• Result – expressed as Index
• Cut-off : Serum : ≥ 0.5
Bronchoalveolar lavage : ≥ 1.5
• Sensitivity : 96.8%
• Specificity : 82.4%
• High negative predictive value :98.3%
• GM in serum ,along with radiological improvement,is used to
monitor treatment.

25. • False positives – Direct cross reaction with antibiotics(β lactams)
- Lipoteichoic acid from Bifidobacterium cross
reacts with the assay ( gut of paediatric age group)
- cross reaction with other fungi (Fusarium
sp.,Histoplasma capsulatum,Alternaria sp.,Paecylomyces
- iv fluids containing GM
- some food items contain GM (popsicle)
- immunosupressive agents
• False negatives – non neutropenic patients
- on anti fungal treatment
• Disadvantages
- Sensitivity decreases following Itraconazole prophylaxis
- Circulating GM is rapidly eliminated by formation of immune
complexes.
Marketers – Platelia Aspergillus
- Pastorex

26. 3.Candidiasis
• Presumptive diagnosis of Invasive candidiasis by Mannan &
Anti-Mannan Assay
 MANNAN & ANTI-MANNAN ASSAY
• Mannan is a highly immunogenic component of cell wall of
Candida species.
• Combined detection of Mannanemia and anti-Mannan
antibodies is useful in diagnosis of Candidemia
• Complemantary to 1,3 β D Glucan assay
• Sandwich ELISA
• Sample – serum ,CSF, bronchoalveolar lavage(pediatric)
• Positive : Mn Ag : ≥0.5ng/ml
anti Mn Ab : ≥10 arbitrary units/ml
• Indeterminate : Mn Ag :0.25-0.5ng/ml
anti Mn Ab : 5-10 arbitrary units/ml

27. • Sensitivity : Mn Ag assay : 58%
Anti Mn Ab assay : 59%
Mn/anti Mn : 83%
• Specificity : Mn Ag assay : 93%
anti Mn Ab assay : 83%
Mn/anti Mn : 86%
• False positives – Candida colonisation
• Disadvantages - Candida mannan is rapidly eliminated
from circulation by formation of immune complexes & by
mannose receptor mediated endocytosis by Kuppfer cells
• Third European Conference on Infections in Leukemia
recommends combined Mn/anti-Mn assay over Mn or
anti-Mn alone in diagnosis of invasive candidemia.
• Marketers – Platelia Candida Antigen (Bio Rad Lab)
- Platelia Candida Antibody (Bio Rad Lab)

28.  Detection of metabolites
D-ARABINITOL
• Large amounts produced by all strains of C.albicans,
C.tropicalis , C.parapsilosis
• Not produced by C.glabrata, C.krusei, Cryptococcus
neoformans
• Serum marker for diagnosis of Candida species
• Therapeutic monitor for invasive candidiasis.
• Sample : serum , urine
SECRETED ASPARTYL PROTEINASE
• Serodiagnostic marker to differentiate between invasive
candidiasis by Candida albicans and Candidal colonization.
• Antigen capture ELISA, inhibition ELISA
Sensitivity – 93.9%
Specificity – 96%
• False positives- aspergillosis
• False negatives- imunocompromised patients (anti SAP Ab)

29. 4.Blastomycosis
• Samples – serum, CSF
• Qualitative assay
• Detection of antibodies to purified yeast phase antigens
• Sensitivity : ̴̴ 85% (negative EIA does not exclude diagnosis
• Specificity : ≥ 95%
• Rapid turnaround time
5.Histoplasmosis
• Screening assay .
6.Coccidioidomycosis
• Screening test

30. LATERAL FLOW ASSAY

31. 1.Cryptococcosis
• Dipstick method
• Screening test and determination of endpoint titres
• Detection of C.neoformans and C.gattii
• Sample – serum , CSF
• LFA titres are higher than LAT titres
• Sensitivity
• Specificity
• False reactions
• Marketers

32. 2.Aspergillosis
• Diagnosis of allergic aspergillosis – detects IgE Ab
• Diagnosis of invasive aspergillosis – detects GM

33. 1,3 β D GLUCAN ASSAY
• Adjunct in diagnosis of IFI
• Pan fungal marker
• Exo antigen- component in majority of fungal cell walls
- Candida sp., Saccharomyces, Trichosporon sp., Sporothrix
sp., Penicillium sp., Fusarium sp., Aspergillus sp.
• Low levels of 1,3 β D glucan (BG) seen in
- Cryptococcus sp.,Histoplasma sp., Coccidioides sp.,
Blastomyces sp., Mucorales.
• Calorimetric assay
• BG triggers coagulation cascade of amoebocyte cells in North
American horseshoe crab(Limulus polyphemus) through
Factor G.

35. • Sample – Serum
- Pus
- CSF
- Bronchoalveolar lavage
• Positive > 80ng/ml
• Use of antifungal drugs does not affect sensitivity of this test
• Sensitivity – 76%
• Specificity – 98%.
• High positive predictive value .- 59%
• Negative predictive value – 97%
• Prevent false reactions by pre treatment with alkali – inhibits
serine proteases

36. USES
• The European Organization for the Research and Treatment of
Cancer and Mycosis Study Group (EORTC/MSG) consensus
has included 1,3β D glucan assay in the diagnosis of IFI.
• Prognostic tool to determine treatment outcome
• Helpful tool in diagnosis of : Pneumocystis jiroveci
Invasive aspergillosis
Invasive candidiasis
• MARKETERS
- Fungitell - Associates of Cape Cod (FDA approved)
- Fungitec G – Seikagaku Biobusiness,Japan
- BGSTAR beta glucan

37. Advantages of serological tests
• To interpret the clinical significance of positive cultures –to
rule out lab contamination
• To identify new isolate when the antibody is demonstrated
against that particular antigen.
• Rapid diagnosis
• Prognostic marker

38. Disadvantages of serological tests
• Kits are expensive which makes continuous monitoring
difficult
• Inability to distinguish between superficial colonization
and deep infection based on the mere presence of
antibodies.
• Antibodies not in detectable levels in the early stage of
disease or immunosuppression.Antigen detection
preferred.
• Detection of macromolecular microbial antigens generally
requires a relatively large microbial burden, which may
limit assay sensitivity.
• Cross reactions – shared antigenicity of several genera and
species of different pathogenic fungi.

39. SUMMARY
• Serology is a useful tool for rapid diagnosis of fungal disease
• Results may be obtained several days before the clinical
symptoms develop.
• Continued screening allows to follow the progress of the
disease, but it is difficult to obtain appropriate specimens.
• Kits are expensive which makes continuous monitoring
difficult.
• Major disadvantage is cross reaction between various
pathogenic fungi. Can be minimised by pretreating sera with
2β mercaptoethanol.
• Tests now in use are 1,3β D Glucan assay, Galactomannan
assay, mannan assay, ELISA, Lateral flow assay.
• No one serological test is confirmatory for the diagnosis of
invasive fungal infections.

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42. THANK YOU