Introduction to Pathogenic Fungi and Superficial Mycoses

Fungi (singular fungus) are eukaryotes. They generally occur in two forms: yeast, which can be round or oval and basically unicellular, but capable of forming long chains called pseudomycelium; and the mold or filamentous. Some pathogenic fungi can be yeast-like inside animal tissue and filamentous in their natural habitat. Also, fungi have a highly developed form of sexual reproduction, but most can also multiply asexually. The natural habitat of a majority of fungi is soil where they perform their primary function in nature, that is, decomposing plant material and recycling the biomass in the ecosystem. However, certain pathogenic fungi are more frequently associated with pigeon or bat excreta. A majority of fungi are harmless to humans and animals. Only a small number of species are known to cause diseases in humans and animals though a majority of plant diseases are caused by fungi. The tissue reaction is usually granulomatous. Fungal infections do not respond to antibacterial antibiotics.

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Treatment of Fungal Keratitis

MEDICAL THERAPY

The antifungal agents available today are mostly fungistatic, requiring a prolonged course of therapy. Although models of Aspergillus and Candida have been established, there are no reliable animal models of Fusarium keratitis. Fungi considered to be ocular pathogens are rarely encountered among the systemic mycoses. Thus, the therapeutic principles valid for systemic fungal infections may not apply to the cornea (O’Day DM 1987). In vitro antifungal sensitivities often are performed to assess resistance patterns of the fungal isolate. However, in vitro susceptibility testing may not correspond with in vivo clinical response because of host factors, corneal penetration of the antifungal, and difficulty in standardization of antifungal sensitivities.

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Molecular-based Diagnostics for Ocular Fungal Infections

POLYMERASE CHAIN REACTION (PCR)

PCR is rapid diagnostic technique to detect the infectious agents even in small volume of samples. PCR is typically used for one of the following scenarios:

1. The patient presents with signs and symptoms that are most likely an infection but a definitive diagnosis cannot be made.

2. When a patient does not respond appropriately to therapy, or

3. For confirmation of a diagnosis when a patient or

4. When the patient's natural history does not coincide with his or her clinical presentation.

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Hyponatraemia: pathophysiology

Hyponatraemia is defined as a serum sodium concentration below the reference interval of 133–146 mmol/ L. It is the electrolyte abnormality most frequently encountered in clinical biochemistry. 

Development of hyponatraemia 

The serum concentration of sodium is simply a ratio, of sodium (in millimoles) to water (in litres), and hyponatraemia can arise either because of loss of sodium ions or retention of water.

- Loss of sodium. Sodium is the main extracellular cation and plays a critical role in the maintenance of blood volume and pressure, by osmotically regulating the passive movement of water. Thus when significant sodium depletion occurs, water is lost with it, giving rise to the characteristic clinical signs associated with ECF compartment depletion. Primary sodium depletion should always be actively considered if only to be excluded; failure to do so can have fatal consequences. 

- Water retention. Retention of water in the body compartments dilutes the constituents of the extracellular space including sodium, causing hyponatraemia. Water retention occurs much more frequently than sodium loss, and where there is no evidence of fluid loss from history or examination, water retention as the mechanism becomes a near certainty. 

Water retention

The causes of hyponatraemia due to water retention are shown in Figure 8.1

Fig 8.1 The causes of hyponatraemia.

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Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 3)

LASIODIPLODIA THEOBROMAE 

Ecology

Worldwide, well known plant pathogen.

Pathogenicity

Lasiodiplodia theobromae is a widespread saprophyte and wound-parasite on a considerable range of hosts in the tropics. It is an important parasite of bananas in storage, causing several forms of fruit-rot. Lasiodiplodia theobromae, a rare cause of mycotic keratitis and endophthalmitis.

Macroscopic Morphology

• Colonies on potato dextrose agar greyish sepia to mouse grey to black, fluffy with abundant aerial mycelium (Fig. 8.38).

• Colonies reverse fuscous black to black.

Microscopic Morphology (Fig. 8.39)

• Conidia initially unicellular, hyaline, granulose, subovoid to ellipsoide-oblong, thick- walled, base truncate; mature conidia 1-septate, cinnamon to fawn, often longitudinally striate, (18-) 20-30 × 10-15 μm.

• The pycnospores are elliptical, at first unicellular and hyaline, becoming brown and 1-septate, sometimes with longitudinal striations, 20-30 × 10-18 μm.

Fig. 8.38: Lasiodiplodia theobromae growth on potato dextrose agar (10 days)

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Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 2)

CANDIDA 

Ecology 

Candida is a genus of yeasts. Clinically, the most significant member of the genus is Candida albicans, which can cause numerous infections (called candidiasis or thrush) in humans and other animals, especially in immunocompromised patients. (Ryan KJ et al, 2004). Various Candida species are members of gut flora in animals, including C. albicans. 

Pathogenicity 

C. albicans is the most common cause of both superficial and systemic candidosis. It is also often present as part of the commensal flora of the mouth, vaginal mucosa and gastrointestinal tract, and may be isolated from these sites in the absence of disease. 

Most episodes of yeast infections in corneal ulcers and other ocular infections are due to various Candida species, predominantly Candida albicans and usually occur in the presence of systemic illness (diabetes mellitus or immunocompromise) or ocular diseases like lid abnormalities or dry eyes) or endogenous endophthalmitis and in patients receiving prolonged topical medications or topical corticosteroids. 

Macroscopic Morphology 

• Candida albicans grows well on potato dextrose agar (Fig. 8.19), Sabouraud’s agar and most routinely used bacteriological media. 

• Convex, entire margin, non-mucoid, smooth texture of cream colored pasty colonies usually appear after 24 - 48 hours incubation at 35-37°C. 

• The colonies have a distinctive yeast smell. 

Microscopic Morphology 

• The round—oval shaped budding cells can be easily seen by direct microscopy in stained or unstained preparations. 

• Candida albicans produces true germ tubes when incubated in serum for 2-3 hours at 37°C (Fig. 8.20). 

• These are parallel-sided tubes which are formed at right angles to the parent cell and are at least twice as long as the parent cell before cross walls are formed.

Fig. 8.19: Cream colored pasty colonies on potato dextrose agar (2 days)

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Macroscopic and Microscopic Characteristics of Ocular Fungal Isolates (Part 1)

ACREMONIUM 

= Cephalosporium (Corda, 1839). 

Pathogenicity 

Acremonium 

Acremonium has been reported as a rare cause of keratitis and endophthalmitis. 

In the literature 17 cases of Acremonium keratitis have been reported between 1965 and  1991. Rosa et al (1994) found 3.2% of Acremonium keratitis in their series and Rodriguez-Areset al reported this as an extremely rare cause of suppurative corneal infection. 

Ecology 

Cosmopolitan, isolated from soil and plant debris. 

MACROSCOPIC MORPHOLOGY 

• The growth rate of Acremonium colonies is moderately rapid, maturing within 5 days. The diameter of the colony is 1-3 cm following incubation at 25°C for 7 days on potato glucose agar. 

• The texture of the colony is compact, flat or folded, and occasionally raised in the center. It is glabrous, velvety, and membrane-like at the beginning. Powdery texture may also be observed. By aging, the surface of the colony may become cottony due to the overgrowth of loose hyphae. 

• The color of the colony is white, pale grey or pale pink on the surface. The reverse side is either uncolored or a pink to rose-colored pigment production is observed (Fig. 8.1). 

MICROSCOPIC MORPHOLOGY

• Acremonium spp. possesses hyaline, septate hyphae which are typically very fine and narrow. Vegetative hyphae often form hyphal ropes. Unbranched, solitary, erect phialides are formed directly on the hyphal tips, the hyphal ropes, or both. The phialides are separated from hyphae by a septum and taper towards their apices. At the apices of the phialides is the hyaline conidia 2-3 × 4-8 μm in size. They usually appear in clusters, in balls or rarely as fragile chains.

• The conidia are bound by a gelatinous material. They may be single or multicellular, fusiform with a slight curve or resemble a shallow crescent. These structural properties of conidia vary depending on the species.

• Acremonium falciforme usually produces crescentic, nonseptate conidia. Sometimes, 2 or 3 celled conidia may also be observed. Acremonium kiliense, on the other hand, has short straight conidia and the conidia of Acremonium recifei are usually crescentic and nonseptate (Figs 8.1 and 8.2).

Fig. 8.1: Acremonium species on potato dextrose agar, 25°C, 7 days

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Identification of Common Ocular Fungal Isolates

TECHNIQUES USED FOR MOULD IDENTIFICATION 

Colony Characteristics

• To evaluate colony characteristics of filamentous fungi, it is necessary to subculture the fungus to the same media that the original colony descriptions are based upon.

• Visual examination of the colony will rapidly reveal important data concerning color, texture, diffusible pigments, exudates, growth zones, aerial and submerged hyphae, growth rate, colony topography, and macroscopic structures such as ascocarps, pycnidia, sclerotia, sporodochia, and synnemata.

Lactophenol Mounts

Prepare a mount of the fungus in lactophenol within the biological safety cabinet.

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Incubation and Processing of Cultures in the Laboratory

Once the specimen has been inoculated on the media by the ophthalmologist, and received in the microbiology laboratory, the plates and tubes or broths must be placed in the appropriate atmosphere and temperature for isolation. Occasionally it is necessary to process specimens that are not set up at the bedside but are submitted to the microbiology laboratory on swabs or in syringes. Given below is the description for processing of specimens for fungal cultures.

INCUBATION REQUIREMENTS 

Conditions (Fig. 6.1) 

Temperature: 25°C to 30°C 

Atmosphere: Sabouraud’s dextrose agar or potato dextrose agar for fungi is incubated in a regular non - CO2 incubator.

Fig. 6.1: Mycological incubator: Maintains temperature at 26°C

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Recommendations for Isolation of Fungi from Ocular Specimens

RECOMMENDED MEDIA FOR FUNGI ISOLATION

Clinical specimens are processed promptly and plated to isolation media as a means to recover fungi that may be causing disease. Media and incubation temperatures are selected to allow for the growth of pathogenic and opportunistic yeasts and fungi.

Isolation Media

The following Table is intended as a guideline for media required for the primary isolation of common isolates in ocular infections. Because some of these sites may be sterile sources and others are nonsterile sources, isolates considered pathogenic differ by site.

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Staining Procedure for Rapid Identification of Fungi

Scrapings from involved ocular site complemented with the appropriate staining method can offer the ophthalmologist circumstantial as well as definitive information concerning the identity of the invading organism.

STAINING METHODS 

Light Microscopy

• KOH

• Gram’s stain

• Giemsa stain

• Grocott-Gomori Methenamine Silver (GMS) stain.

Fluorescent Microscopy

• Calcofluor white staining

• KOH-Calcofluor white procedure.

KOH mount

Principle: Potassium hydroxide is used as a mounting fluid for visualization of fungal filaments as it helps in the clearance or lyses of all surrounding tissues. In addition, Acanthamoeba cysts and Nocardia filaments can also be visualized.

Preparation of 10% KOH

• Approximately 1 gm (8 pellets) of KOH is weighed

• It is dissolved in 10 ml of distilled water

• One drop of 10% glycerol is added

• Fresh stock should be prepared every week

Can be kept at room temperature in a dropper bottle like a penicillin bottle (Fig. 4.1).

Fig. 4.1: KOH pellets and preparation of 10% KOH

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Ocular specimen collection

Standard procedures followed for ocular specimen collection, culture isolation and identification of the organisms are described below.

SPECIMEN COLLECTION KIT TO BE KEPT IN THE OPHTHALMOLOGIST OFFICE

The following are the items that must be available for collecting the corneal specimens.

• Culture plates (blood agar, potato dextrose agar)

• Sterile Kimura’s spatula or scraper

• Topical anesthetic agents

• Bunsen burner

• Clean glass slides

• Clean cover slips.

Corneal Scraping

Corneal scraping is performed under aseptic conditions by an ophthalmologist using a sterile Kimura spatula. The procedure is performed under magnification of a slit lamp or binocular loupe following instillation of topical anesthetic agents such as 0.5% proparacaine or 4% lignocaine.

Material obtained from scraping the leading edge and the base of each ulcer specimen are inoculated directly onto sheep blood agar, potato dextrose agar (PDA) and Brain Heart Infusion broth (BHI) without gentamycin sulphate. The material from the corneal scraping is also smeared and labeled onto slides in a thin, even manner to prepare a 10% KOH wet mount and Gram staining. In cases of suspected actinomycetes keratitis Kinyoun’s metho of acid fast staining is performed (Fig. 3.1). Corneal scrapings collected by an opthalmologist by using an ophthalmic microscope in shown in Figures 3.4A to F.

In some of the patients, the ulcer may be predominately in the deeper stromal lesion, with inflammatory outpourings in the anterior chamber. In such a case, a paracentesis is made using a 26 gauge needle mounted on a 2cc plastic disposable tuberculin syringe (Figs 3.2A and B).

Fig. 3.1: Materials used for collecting specimens from corneal ulcers

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Clinical Presentation of Fungal Keratitis

The early lesions of fungal keratitis are quite characteristic. The symptoms are less than what the size would warrant. The symptoms are also less than that of bacterial keratitis of similar size. Some of the characteristic clinical manifestations include, dry raised surface, feathery margins, satellite lesions and posterior corneal abscess. Of these, feathery margins are very typical of fungal corneal ulcers (Figs 2.1 to 2.21).

Fig. 2.1: An early fungal ulcer presenting with very mild congestion and few symptoms. In fungal keratitis the signs are disproportionately higher than the symptoms

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