PEARLS OF LABORATORY MEDICINE
Dennise E. Otero Espinal MD
Assistant Director of Clinical Pathology at Lenox Hill Hospital
DOI: 10.15428/CCTC.2019.304881
© Clinical Chemistry Objectives
• Name methods for mold identification
• Describe the characteristics of hyaline molds
• Discuss some of the most commonly Mucorales fungi isolated in the laboratory
2 Methods of Identification • Direct visualization • Slide prepared before setting fungal cultures • Calcofluor White fluorescent stain o Non- specific 1 Hyaline mold stained with Calcofluor White fluorescent stain. Photo credit: Melinda Wills • Histology o Hematoxilin & Eosin stain (H&E) o Gomori Methenamine Silver stain (GMS) o Periodic acid–Schiff (PAS)
o Non-specific 2 Rhizopus spp. - H&E, 60x 3 Methods of Identification
• Culture • Colony morphology • Lactophenol cotton blue stain • Media types:
o Non-selective
o With antibiotics
o With cycloheximide • MALDI-TOF MS • DNA sequencing Penicillium spp., lactophenol cotton blue stain, 60x
4 Hyaline Molds • Rapid growth
• Thin, regularly septate hyphae
• Acute angle branching septations (blue arrows)
• Aspergillus spp., Fusarium spp., and Scedosporium spp. may
be indistinguishable by Hyaline mold, H&E stain, 60x histology
5 Hyaline Molds - Aspergillus fumigatus Complex
• Most common cause of invasive aspergillosis, allergic aspergillosis, fungal sinusitis, and aspergilloma (fungus ball) 1
• Fast growing blue-green colonies with white borders (image 1), white to tan on reverse
• Dome or flask-shaped vesicle with uniseriate phialides covering 2/3 of the vesicle (red arrows image 2)
2
A. fumigatus complex - Lactophenol cotton blue stain, 60x
6 Hyaline Molds - Aspergillus flavus Complex
• Second most common cause of invasive aspergillosis • Producer of aflatoxin
• Fast growing, yellow-green 1 colonies, yellowish on reverse (image 1)
• Rough or spiny conidiophore, may be hard to see (red arrow image 2) • Globose vesicle covered by uniseriate or biseriate phialides 2 A. flavus complex – Lactophenol cotton blue stain, 60x
7 Hyaline Molds - Aspergillus niger Complex
• Cause of aspergilloma (fungus ball) and otitis externa
1 • Fast growing black colonies, white on reverse (image 1)
• Globose vesicle with biserate phialides and brown conidia (image 2 2) A. niger complex - Lactophenol cotton blue stain, 60x
8 Hyaline Molds - Aspergillus terreus Complex • Can cause disseminated disease 1 • Resistant to amphotericin B
• Fast growing cinnamon- brown colonies, yellow on reverse
• Upper half of the dome- shaped vesicle has biserate phialides (red arrow) 2 A. terreus complex - Lactophenol cotton blue stain, 60x 9 Hyaline Molds - Penicillium spp.
• Most often considered to be a contaminant • Talaromyces marneffei (Penicillium marneffei) 1 • Fast growing, powdery blue-green colonies (image 1)
• Branched and unbranched conidiophores with clusters of phialides with round conidia in chains (image 2)
2 Penicillium spp. - Lactophenol cotton blue stain, 60x
10 Hyaline Molds - Paecilomyces spp. • Considered a contaminant • May be a cause of keratitis
• Usually fast growing, flat, yellow-brown colonies, with off- white reverse
• Branched conidiophores with clusters of phialides with delicate tapering ends and oval conidia in chains Paecilomyces spp. – Lactophenol cotton blue stain, 60x
11 Hyaline Molds – Penicillium Vs. Paecilomyces
Penicillium Paecillomyces
Blue-green colonies Yellow-brown colonies
Blunt phialides Long tapering phialides
Round conidia Oval conidia 12 Hyaline Molds - Fusarium ssp.
• Can cause a wide array of infections, from nail to disseminated disease (in immunocompromised patients)
• Fast growing wooly colonies • White, cream, pink, or purple • Light or deeply colored reverse
• Produces micro and macroconidia • 1-2 celled microconidia • Curved macroconidia (banana or sickled shaped), septate, Fusarium spp. - Lactophenol cotton blue stain, 60x usually in clusters
13 Hyaline Molds – Acremonium spp. • Cause of white-grain mycetoma, keratitis, and nail infection
• Slow growing rose to white colonies
• Clusters of single cell conidia at the end of narrow phialides (red arrow), usually in masses Acremonium spp. - Lactophenol cotton blue stain , 60x Photo credit: Melinda Wills 14 Mucormycosis
• Caused fungi from the Mucorales order • Rhizopus spp., Mucor spp., Lichtheimia (previously Absidia) spp., Rhizomucor, and Apophysomyces spp and others rarely
• Zygomycetes - obsolete term
• Cause of rhinocerebral, pulmonary, cutaneous, and systemic invasive disease • Diabetes, iron overload, immune suppressed
15 Mucorales
• Rapidly growing mycelium
• Pauciseptated, ribbon-like or broad hyphae
• Wide-angle branching Rhizopus spp. - H&E stain, 60x • Histology
16 Mucorales - Rhizopus spp.
• Most common cause of mucormycosis
• White, cottony, fast growing colonies that darken with age. White to pale grey or brown reverse • Characterized by presence of rhizoids • Root like structures (red arrow)
Rhizopus spp. - Lactophenol cotton blue stain, 60x • Multispored, spherical sporangia • Unbranched sporangiophores directly connected to rhizoids 17 Mucorales - Mucor spp.
• Less common cause of mucormycosis
• White-yellow to grey, cottony, fast growing colonies that 1 darken with age (Image 1). Reverse is white
• Hyaline sporangiophores • Multispored, spherical sporangia • No rhizoids (red arrow image 2) • Identification of exclusion
2 Mucor spp. - Lactophenol cotton blue stain, 60x 18 Mucorales
• Differential characteristics between Lichtheimia, Rhizomucor, and Apophysomyces
Genus Sporangiophore Apophysis Columella Sporangium Rhizoids Apophysomyces Unbranched Bell- Dome Pyriform Present grey-brown shaped, not shaped prominent Lichtheimia Branched Conical, not Dome Pyriform Primitive, hyaline prominent shaped present Rhizomucor Branched Absent Round Globose Present, brown few
19 References
1. Chen SCA, Sorrell TC, Meyer W. Aspergillus and Penicillium. In: Jorgensen JH, Pfaller MA, Carroll KC et al., eds. Manual of Clinical Microbiology. 11th ed. Washington DC: ASM; 2015: 2030- 2056.
2. Zhang SX, O’Donnell K, Sutton DA. Fusarium and Other Opportunistic Hyaline Fungi. In: Jorgensen JH, Pfaller MA, Carroll KC et al., eds. Manual of Clinical Microbiology. 11th ed. Washington DC: ASM; 2015: 2057-2086.
3. Garcia-Hermoso D, Alanio A, Lortholary O, et al. Agents of Systemic and Subcutaneous Mucormycosis and Entomophthoromycosis. In: Jorgensen JH, Pfaller MA, Carroll KC et al., eds. Manual of Clinical Microbiology. 11th ed. Washington DC: ASM; 2015: 2087-2109.
4. Love GL, Ribes JA. Color Atlas of Mycology: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists; 2018.
5. Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev 2011;24:247-80.
6. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev 2000;13:236-301.
7. Mendoza L, Vilela R, Voelz K, Ibrahim AS, Voigt K, Lee SC. Human Fungal Pathogens of Mucorales and Entomophthorales. Cold Spring Harb Perspect Med. 2014;5(4):a019562. 20 Disclosures/Potential Conflicts of Interest
Upon Pearl submission, the presenter completed the Clinical Chemistry disclosure form. Disclosures and/or potential conflicts of interest:
. Employment or Leadership: No disclosures . Consultant or Advisory Role: No disclosures . Stock Ownership: No disclosures . Honoraria: No disclosures . Research Funding: No disclosures . Expert Testimony: No disclosures . Patents: No disclosures
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