---

Pentacam Criteria for Diagnosing Keratoconus

---

1. Topometric / KCN Staging (TKC) - Automated Classification

---

2. Belin/Ambrosio Enhanced Ectasia Display (BAD-D) - Most Sensitive Screen

The 5 "D" Parameters (SD from normal mean):

Color coding:

• White = < 1.6 SD from mean (normal)
• Yellow = ≥ 1.6 SD from mean (suspicious)
• Red = ≥ 2.6 SD from mean (abnormal)

The Final "D" (BAD-D) Score:

• Composite regression index combining all 5 D-values against a validated normal/keratoconic database
• BAD-D > 1.6: Suspicious
• BAD-D > 2.6: Highly suggestive of ectasia / keratoconus

4 Additional Reported Parameters:

• ARTmax (Ambrosio Relational Thickness maximum) - thinnest pachymetry divided by the maximum pachymetric progression index
• Anterior elevation at thinnest point
• Posterior elevation at thinnest point
• Kmax (maximum keratometry)

---

3. Elevation Map Criteria (Manual Best-Fit Sphere Analysis)

Using Fixed 9 mm zone BFS:

Using Fixed 8 mm zone BFS:

The posterior elevation map is more sensitive than anterior for early/subclinical keratoconus detection. In keratoconus eyes, average posterior elevation change is ~40-46 µm vs. ~2.5 µm in normals (p<0.0001).

---

4. Pachymetric Criteria

---

5. Curvature / Keratometry Criteria

---

6. Belin ABCD Grading System (Staging)

• A = Anterior radius of curvature (3 mm zone centered on thinnest point)
• B = Posterior radius of curvature (3 mm zone centered on thinnest point)
• C = Thinnest corneal pachymetry
• D = Distance-corrected visual acuity (BDVA, manually entered)
• Each parameter is graded independently - a patient can be Stage II-A but Stage III-B

---

7. Summary: Red Flags on Pentacam for KCN Diagnosis

Key principle: No single parameter is diagnostic alone. The Pentacam's strength is the combination of posterior elevation + pachymetric progression + curvature, especially in subclinical/forme fruste cases where anterior topography may appear normal.

---

Stains Used in Corneal Ulcers - Complete Reference

Quick Master Table: Stain vs. Organism

---

1. BACTERIAL KERATITIS

Gram Stain (PRIMARY stain)

• What it shows: Differentiates organisms by cell wall into Gram-positive (purple/violet) and Gram-negative (pink/red)
• Smear positivity: ~60-75% in bacterial keratitis; ~25-35% in mixed studies
• Key findings:
  • Gram-positive cocci in clusters - Staphylococcus
  • Gram-positive cocci in chains - Streptococcus
  • Gram-negative rods - Pseudomonas, Moraxella, Enterobacteriaceae
  • Branching Gram-positive filaments - Nocardia (also weakly acid-fast)
  • Small pleomorphic Gram-negative coccobacilli - Moraxella, Haemophilus
• Counterstain: Safranin (Gram-negative = pink); Crystal violet (Gram-positive = purple)
• Limitation: 65% of smears may be negative, especially with prior antibiotic use

Giemsa Stain

• What it shows: Morphology of organisms AND host cell response; stains nucleus and cytoplasm differentially
• Key findings in bacteria:
  • Intracellular organisms: Chlamydia (basophilic intracytoplasmic inclusions)
  • Inflammatory cells (PMNs, macrophages) showing acute vs. chronic reaction
  • Bacteria stain blue-purple; background cells stain pink/blue
• Advantage over Gram: Better for visualizing cell morphology, good for detecting mixed infections; Giemsa may be better for fungal identification than Gram in some studies

Ziehl-Neelsen Acid-Fast Stain

• Used for: Mycobacterium (non-tuberculous, e.g., M. chelonae, M. fortuitum) and Nocardia
• Principle: Carbol fuchsin + heat → decolorized with acid-alcohol → counterstained with methylene blue
• Findings: Acid-fast organisms stain bright red against blue background
• Nocardia: Partially acid-fast (modified ZN with 1% H₂SO₄ instead of 3% HCl)
• When to suspect: Post-LASIK keratitis with gray-white cracked-windshield infiltrate, atypical slow-growing ulcer

---

2. FUNGAL KERATITIS

KOH Wet Mount (PRIMARY rapid stain for fungi)

• Concentration used: 10% KOH (10-20% used clinically)
• Mechanism: KOH dissolves keratin and cellular debris, leaving fungal elements intact
• Preparation: Drop of 10% KOH + scraping on slide → cover slip → examine under 10x and 40x
• Key findings:
  • Filamentous fungi (e.g., Fusarium, Aspergillus): Branching septate hyphae (double-walled, branching filaments visible)
  • Mucor/Rhizopus (rare): Broad aseptate/pauciseptate hyphae
  • Candida: Pseudohyphae + budding yeast cells
• Sensitivity: ~90% for filamentous fungi
• Specificity: >90% in experienced hands
• Important: Use low-intensity light for contrast - high intensity washes out unstained fungi

Calcofluor White (CFW) / KOH + CFW (BEST stain for fungi)

• Mechanism: Fluorescent dye that binds to chitin and cellulose in fungal cell walls
• Findings: Fungi fluoresce bright apple-green or blue-white under UV/fluorescent microscopy
• Combined KOH+CFW: Most sensitive combination - >95% sensitivity for filamentous fungi and Acanthamoeba
• Key advantage: Can detect small amounts of fungal material invisible on KOH alone
• Also detects: Acanthamoeba cysts fluoresce brightly

Gomori Methenamine Silver (GMS) / Modified Grocott GMS

• Mechanism: Silver impregnates fungal cell wall polysaccharides
• Findings: Fungi stain black/dark brown against green counterstain
• Used mainly on: Tissue sections (corneal biopsy) and culture smears
• Advantage: Extremely sensitive - identifies even non-viable fungal elements in tissue

Periodic Acid-Schiff (PAS)

• Mechanism: Oxidizes fungal cell wall polysaccharides → Schiff reagent → magenta color
• Findings: Fungi stain bright magenta/pink-red against blue/green counterstain
• Used on: Tissue sections (corneal biopsy specimens)
• Also stains: Goblet cells, basement membranes (so background can be noisy)

Lactophenol Cotton Blue (LPCB)

• Used for: Identification of fungal morphology in culture (not directly on scrapings)
• Findings: Stains fungal structures blue - allows identification of conidia, hyphae type, sporulation
• Helps identify: Aspergillus vs. Fusarium vs. Candida species in culture

Acridine Orange

• Fluorescent stain binding nucleic acids
• Bacteria: Stain orange-red; background tissue = green
• Fungi: Stain yellow-green
• Advantage: Rapid, can detect bacteria AND fungi on the same slide; useful when Gram stain is equivocal

---

3. ACANTHAMOEBA KERATITIS

Calcofluor White (CFW) - PRIMARY / MOST SENSITIVE stain

• Mechanism: Binds chitin in Acanthamoeba cyst walls
• Findings: Cysts fluoresce bright blue-white or apple-green under fluorescence microscopy
• Appearance: Double-walled cysts with polygonal/stellate inner cyst wall (endocyst visible)
• Sensitivity: Best sensitivity for cysts among all stains
• Limitation: Requires fluorescence microscope; cannot distinguish cyst types

Gram Stain

• Findings: Cysts and trophozoites may be visible but poorly defined
• Trophozoites: ~15-45 µm, amorphous, with Gram-variable staining
• Cysts: ~10-25 µm, double-walled, may appear as clear round structures
• Sensitivity: Lower than CFW; ~79-86% in studies - better for trophozoites than cysts

Giemsa / Giemsa-Wright Stain

• Findings:
  • Trophozoites: Stain with pale blue cytoplasm, darker blue-purple nucleus with prominent karyosome (nucleolus)
  • Cysts: Appear with double walls; inner cyst (endocyst) stains more darkly
• Key feature to identify: The distinctive large karyosome in trophozoite nucleus

Hematoxylin & Eosin (H&E)

• Used on: Tissue sections (biopsy, keratoplasty button)
• Findings: Cysts appear as clear, double-walled round structures in stroma with surrounding inflammatory cells
• Trophozoites: Poorly identified; cysts better defined

Periodic Acid-Schiff (PAS)

• Findings: Cyst wall stains magenta (due to glycoprotein content of cyst wall)
• Used on tissue sections - helps confirm cysts in histopathology

KOH Wet Mount

• Can detect Acanthamoeba cysts (double-walled, wrinkled appearance)
• Sensitivity: Lower than CFW but useful as a rapid screen
• Acanthamoeba cysts seen as double-walled, polygonal refractile structures

---

4. SPECIAL/ADDITIONAL STAINS SUMMARY

---

5. SENSITIVITY COMPARISON (from NIH/Esterberg data - South India tertiary center)

---

6. PRACTICAL CLINICAL PROTOCOL (Per MSO Series / EyeWiki)

1. Gram stain - first-line for bacteria
2. KOH 10% wet mount - first-line for fungi
3. Giemsa stain - morphology, mixed organisms, Chlamydia

• Fungal → Add GMS or PAS (if biopsy), Calcofluor white
• Acanthamoeba → Calcofluor white (most important), Giemsa for trophozoite morphology
• Mycobacteria/Nocardia → ZN acid-fast stain, Auramine O
• Viral → Tzanck smear (Giemsa), PCR preferred

---