SEROLOGICAL TESTS IN

MYCOLOGY

A.SIVARANJINI

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

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.

• 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

FUNGAL PATHOGENS OF MEDICAL

IMPORTANCE OPPORTUNISTIC• Candida species• Aspergillus species• Cryptococcus species• Pneumocystis jiroveciTRUE PATHOGENS• Coccidioides immitis• Paracoccidioides brasiliensis• Histoplasma capsulatum• Blastomyces dermatitidis

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.

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)

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)

DIAGNOSIS1.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

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 negatives3.Diagnosis of Paracoccidioidomycosis• Paracoccidioides brasiliensis• Sensitivity – 84%• Specificity – 81%• False positives – Aspergillosis , Histoplasmosis.

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

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

3. Candidiasis• Detection of Anti- Mannan antibodies4.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

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

• 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

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

COMPLEMENT FIXATION TESTThe 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

Click icon to add picture

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

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

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

ENZYME LINKED IMMUNO SORBENT ASSAY

1.Cryptococcosis• Sample • Sensitivity• Specificity

2.Aspergillosis • Allergic aspergillosis - detection of IgE Ab• Invasive aspergilllosis - GALACTOMANNAN(GM) assay

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.

• 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

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

• 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)

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)

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 time5.Histoplasmosis• Screening assay .6.Coccidioidomycosis• Screening test

LATERAL FLOW ASSAY

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

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

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.

• 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

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

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

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.

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.

• REFERENCES• Jagdish Chander.Mehta Publishers.Textbook of Medical

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