This document discusses serological, molecular, and histopathological techniques for diagnosing fungal infections. It covers techniques for detecting antibodies, antigens, metabolites, and nucleic acids from fungi like Candida, Aspergillus, Cryptococcus, Histoplasma, and Blastomyces. Molecular techniques discussed include electrophoretic karyotyping, hybridization, PCR, RFLP, and sequencing. Histopathological diagnosis using stains like GMS, PAS, H&E are described. The document provides detailed information on diagnostic methods for various medically important fungi.

1. Serological, Molecular & Histopathological
Diagnosis Of Fungal Infections
Dr. Kanwal Deep Singh Lyall
M. D. Microbiology

2. Mycoserology
• Candidiasis
• Aspergillosis
• Cryptococcosis
• Histoplasmosis
• Blastomycosis
• Coccidiodomycosis
• Paracoccidioidomycosis

3. Candidiasis
• Disseminated candidaemia (DC)
• Specific clinical features lacking
• Blood cultures often negative
• Single positive blood culture does not confirm Dx
• Early initiation of therapy critical in reducing mortality
• Candida antibodies
• Cand-tec latex agglutination assay
• Mannan
• Protease
• Enolase
• Hsp90 metabolites

4. Candida antibodies
• Several kits available
• Intrinsically flawed
• Antibodies occur in superficially infected
• Patients with worsening DC – fail to produce Abs
• Except in endocarditis
• In DC good AB response = marker of recovery
• Lack of AB = poor prognosis

5. Cand-Tec latex agglutination assay
• Gentry et al. – Ramco Inc., Houston Tx
• Reverse passive latex agglutination assay
• Latex particles sensitized with rabbit Abs against heat-killed
blastoconidia
• Target Ag not yet characterized (heat labile, proteinaceous)
• Sensitivity 33-71%
• A titre of 1: 4 = + (1:8 more sign.)
• False -ve = C. krusei, C. lusitaneae, C. parapsilosis (not readily
detected)
• False +ve = Aspergillus spp. & C. neoformans, RA factor +ve sera,
• Helpful in monitoring therapy, as titres fall with recovery & rise
with relapse

6. Mannan
• CW mannanoprotein = carb. Homopolymer mannose + small
ammount of protein + phosphate
• Resitant to boiling, proteases, acidic pH
• In serum found as immune complexes – dissociation required –
proteases, boiling, heating , extreme pH etc.
• EIAs, double Ab sandwich EIAs–high specificity, sensitivity 23-
90%
• Relative spp. Specificity well documented
• Detection of Ab agaisnt mannan also tried
• Reverse passive latex agglutination kits – using polyclonal &
monoclonal Abs – inferior to EIAs – sens 71% & spec 98% ( titre
1:8 sign.)

7. Protease
• Circulating secreted acid protease Ag & specific Abs described
• Protease facilitates epidermal invasion
• Assay detecting urinary protease described in rabbits, little
human data available
• Diff. spp. – antigenically distinct proteases
• Some spp. (C. krusei) – no protease
• Therefore – test complicated & of limited value

8. Enolase
• Abundant cytoplasm enzyme-glycolytic pathway
• All Candida spp. Produce enolase
• Liposomal sandwich assay – sera passed through NC
membrane to which IgA murine monoclonal Ab against
enolase adsorbed – bound enolase detected by specific rabbit
polyvalent antiserum – f/b goat anti-rabbit Ig labeled with
liposome detector particles coating a red dye
• Sensitivity 75%, specificity 96%

9. Hsp90
• Heat shock proteins k/a stress proteins
• A 47kd Ag, subcomponent of Hsp90 – found in sera of patients
of DC
• Detected using rabbit antiseum to Candida spp. With dot
immunobinding assay

10. Metabolites
• Most Candida spp. (except C. glabrata & krusei) – metabolite D-
arabinitol
• Meaured using gas-liquid chromatogrphy – cumbersome & limited
availability
• Enzymatic fluorometric metohod- oxidation of arabinotol by
arabinotol dehydrogenase – concominant reduction of NAD –
NADH – initial rduction rate – proportional to serul arabinotol –
measured using spectrofluorometer
• Commercially available kits – sen.50% , specificty 91%

11. Aspergillosis
• Detection of Ab has a role, no role of Ag & DNA detection –
ABPA & aspergilloma - EIAs, immunoblot assay
• RAST , Skin prick test,– IgE med. Hypersensitivity – A.
fumigatus
• RIA, EIAs, LAT – circulating galactomannan

12. Cryptococcosis
• Meningitis, pneumonia, fever d/t cryptococcaemia
• Capsular polysaccharide antigen – heat & pronase stable – allow
dissociation from bound complexes – LAT – CSF/serum
• CALAS (meridian), Crypto-LA (international biological labs), Pastorex
cryptococcus (Sanofi-diagnostics)
• A heat stable CW component of Trichosporon beigelii - cross Rx with
cryptococcal polysaccharide
• Technical false +ve d/t talc from latex gloves in CSF, immersion of
platinum loop into CSF, wasshing slides with certain detergents etc.
• False neg. – prozone like phenomona, AIDS patients, dry variants,
localized cryptococcosis.

13. • EIAs (meridian)- uses polyclonal capture system to bind cap.
Polysacch-visualized with peroxidase-labelled MABs – sens –
99%, spec – 97%
• Detection of culture filtrate antigens (exantigens) – capsulated
& non-capsulated Ags – using murine MABs

14. Histoplasmosis
• Diamorphic fungus – H. capsulatum – common complication in AIDS
• Histoplasmin skin testing
• CFT, immunodiffsion, LAT – Ab detection
• Hsitoplasmin LA test (immuno-mycologics) – detects IgM – double
diffsuion test – pt’s serum & soluble Ags placed in wells cuts in agarose
or Cleargel – allowed to diffuse outward – precipitation lines at Ag-Ab
combination- detects IgG (-ve in 1st 3-6wks) – can detect H & M Ags
• CFT – C’ fixed by Ag-Ab complex – no hemolysis – more sens. Than ID
• RIA – detection of polysacch. in urine, BAL

15. Blastomycosis
• Diamorphic fungus – Blastomyces dermatidis
• Commercially available kits – CFT, ID –meridian, Gibson,
Lexington diagnostics etc. – detection of A antigen – low
sensitivity
• An indirect EIA – purified A antigen – sens. 80% , spec. 98% -
titres 1:32 or greater

16. Coccidioidomycosis
• Diamorphic fungus – Coccidiodes immites
• SeroDx – detection of Abs to 2 main Ags : tube precipitin (TP) & CF
Ags (coccidioidin) – mainly IgG Ab
• LAT – Immuno-Mycologics – TP-coated latex particles to detect
agglutinating Abs – simple, rapid – high false +ve – confirmation by ID
is essential
• IDTP – detects IgM, IDCF – detects – IgG – serum/ CSF samples
• EIAs – IgM & IgG – commercialy available – Meridian – sens – 94.8%
& spec. 98.5%

17. Paracoccidioidomycosis
• Diamorphic fungus Paracoccidioides brasiliensis
• ID, CIE, (sens. 91.3%, 95.6% respectively, spec. 100%)
• ID commercially available by immuno-mycologics
• Measurement of Ab – usefull in diagnosis and prognosis – titres fall
with treatment

18. Molecular Techniques
• Electrophoretic karyotype analysis
• Hybridization
• PCR
• RFLP
• Sequencing

19. Electrophoretic Karyotype Analysis
• Banding pattern visualized on gel
• Each band = large chromosomal DNA
separated by PFGE
• Electric current switched inversely over a short
period
• Disentangling of long DNA strands
• Separation by size

20. Cells solidified with agarose gel of low melting point
Digestion of CW with enzymes
Spheroblasts generated are lysed
DNA released by detergent & protease K & washed
Gel plugs containing DNA are loaded on gel
Electrophoresis
Staining with ethidium bromide
Photgraphed under UV light
Identification

21. • Candida species
• Candida albicans
• C. neoformans
• Coccidioides immitis
• Malassezia furfur

22. Hybridization
• Ndna or mtDNA – 2 strands – denaturation by Tm & alkaline
conditions
• annealing or hybridization (2strands of diff origin) at lower
temp
 Determination of base sequence homologies
 Detection of target sequences with probe
 Southern blot hybridization
 Binding the primer to the template DNA in PCR

23. Determination of base sequence homologies
• DNA fixed on membrane→denaturated & hybridized with another
labelled single stranded DNA→ unhybridized DNA washed out→hybrid
DNA detected
• Hypochromic shift→dsDNA→absorbance at 260nm ↑es rapidly & ↓es on
reassocitaion→Hyperchromic to hypochromic shift
• Slow shift = DNA of diff. homology
• Fast shift = homologous DNA
• Time measured = level of sequence homology
• Sporothrix schenckii
• Dermatophytes

24. Detection Of Target Sequence With Probe
• Short single strand of DNA - complimentary to target
- seek out their complimentary sequence – hybridize
rapidly to make more chemically stable dsDNA
• Aspergillus fumigatus specific probe

25. Southern Blot Hybridization
• High-molecular-weight DNA strands cut into smaller
fragments
• Electrophoresed on agarose gel
• Separation by size
• DNA moved to membrane
• Baked for permanent attachment
• Exposed to labeled hybridization probe
• Excess probe washed away
• Pattern visualized on x-ray film or by development of color on
the membrane

26. • Candida albicans
• Saccharomyces cerevisiae
• Histoplasma capsulatum
• Cryptococcus neoformans

27. @ 94–98 °C x 20–30 seconds
@ 50–65 °C x 20–40 seconds - primers
@ 75–80 °C (72 °C) - enzyme taq
polymersae
•1 PCR cycle = 1 molecule of
DNA= 2 mol of DNA = 4 strands
of DNA
• Standard amplification = 25-35
cycles = billion copies of original
product
PCR

28. • Candida albicans
• Candida tropicalis
• Candida parpsilosis
• Aspergillus spp.
• Hortaea werneckii
• Exophialla dermatidis
• Malassezia spp.

29. Restriction Fragment Length Polymorphism
• Compare degree of base sequence similarity indirectly
• Long dsDNA digested with 1 or more restriction enzymes
• Fragments of diff length produced – separated by agarose gel
electrophoresis
• Transferred to membrane by southern blotting
• Staining with ethidium bromide
• Or
• Banding pattern obtained on gel is compared
• If DNA sequence diff. – length of fragments diff.
• Used for typing and estimating sequence similarities

30. Sequencing
• To determine 1º structure of an unbranched biopolymer
• Results in symbolic linear depiction k/a sequence
• Summarizes atomic-level structure of sequenced molecule
• Chain terminator sequencing (Sanger sequencing) – synthesis
using DNA of interest as template
• Pyrosequencing – fragments prepared by cutting long DNA of
interest
• Diff. lengths of fragments ending with a specific base
(A,G,T,C)
• Solving taxonomic, phylogenetic & epidemiological problems

31. • Sporothrix schenckii lies phylogentically in
genus Ophiostoma
• Candida spp. & B. dermatidis have been
investigated phylogenetically

32. Histopathological Diagnosis
• Fixed histopath. Material may be only material
• No viable fungal elements
• Rhinosporodosis, lobomycosis, P. carini – culture conditions
not defined – only histopath diagnosis
• Many opportunistic fungi – detection in tissue & confirmation
of invasion = histopath.
• Eg. Aspergillus, penicillium, rhizopusin sputum – not
confirmed pulm. Infection
• Rapid, relatively inexpensive

33. Disadvantages
• Speciation possible only when monotypic fungal genus
involved or when only 1 species in genus is known to be
pathogenic
• Unusual fungal morphology
• Taxonomically diverse etiological agent having similar
morphology in tissue
• Unusual staining

34. Stains used
• Haematoxylin and eosin (H&E)
• Special stains
 Gomori’s methenamine silver (GMS)
 Gridley’s fungus (GF)
 Periodic acid-Schiff (PAS)
 GMS with H&E
• Mucin stains
 Mayer’s mucicarmine
 Southgate’s mucicarmine
 Alcian blue
• Melanin stain –modified Fontana-Masson

35. H&E
• Innate color of fungus determined
• Pink to pikish blue
• Better observation of tissue response
• Revels the Splendore-Hoeppli phenomena
Limitations
• Many fungi poorly stained or not stained at all
• Inadequate to screen fungal elements
• Does not stain filaments of actionomycetes, nocardia and
streptomyeces

39. Special stains
• Best for detecting fungi
• Not adequate to study tissue response
• Fungal CW stained well but internal elements obscured
• Principle – adjacent hydroxyl groups of complex
polysaccharides in fungal CW – oxidized to aldehydes by
chromic & periodic acid
• GMS, GF, PAS & GMS +H&E

40. GMS
• Preferred over other stains
• Best for tissue sections
• Aldehydes reduce silver nitrate – silver gets deposited
wherever aldehydes +nt
• Brownish-black coloration of viable & NV fungal elements
• Light green or light yellow background
• Nocardia, actinomycetes, mycobacteria, cysts of P. carinii,
free living amoeba etc. - stain well
Limitations
• Over-staining – host cells mimic fungal cells
• Innate color not determined

41. vascular invasion by Aspergillus with associated thrombosis in
the lung (GMS stain, original magnification × 400).

42. • Phycomycosis - broad, pleomorphic hyphae , thin walled, delicate and pauciseptate.
• The hyphae are often twisted, folded and wrinkled and have the appearance of
folded ribbons weaving through cells.
• Hyphae are best demonstrated with GMS as shown in this photograph.

43. GF
• Aldehydes react with schiff reagent – magenta
color
• Purplish red on yellow background
• Fungi readily detected
• Morphologic details of fungi visible
Limitations
• Innate color masked
• Dead fungi may not be stained

44. PAS
• Aldehydes react with schiff reagent – magenta
color
• Black brown on light-green background
• Readily detects most fungi
Limitations
• Innate color masked
• Internal details may be obscured
• Does not detect tissue response
• Does not stain dead fungi

45. • Disseminated histoplasmosis of the large bowel. Periodic acid-Schiff (PAS)
light green stain (250) revealing myriads of small budding yeasts within the
lamina propria. An added benefit of examining PAS-light green stained
sections is that the underlying architecture of the specimen can easily be
observed.

46. GMS with H&E counterstain
• Special stains – innate color masked
• Pigmented fungi – not determined
• Also inadequate study of host reaction
• H&E used as C/S
• Detection of fungus + evaluation of inflammatory response
• Optimal for photomicrography
• Best when single slide submitted
Limitations
• Innate color not determined
• To improve - 1st stain with H&E, then GMS-H&E

48. Mucin stains
• Mayer’s mucicarmine, Southgate’s mucicarmine, Alcian blue
• Stain MPS capusule of C. neoformans
• Differentiates C. neoformans from similar fungi
• limitations
• Nonspecific for C. neoformans
• Capsule –ve C. neoformans may not stain
• May stain Rhinosporidium seeberi & some cells of
Blastomyces dermatidis

50. Melanin stain –modified Fontana-Masson
• CW of C. neoformans – melanin like, silver reducing
substances
• React +vely with mod. Fontana-Masson stain for melanin
• Reaction not dependent on mucinous capsule – detects capsule
–ve variants
Limitations
• May stain CW of Sporothrix schenckii & immature spherules
of Coccidiodes immitis

51. THANK YOU