Point of care testing

The methods for measuring some bio- logical compounds in blood and urine have become so robust and simple to use that measurements can be made away from the laboratory – by the patient’s bedside, in the ward sideroom, at the GP’s surgery, at the Pharmacy or even in the home. Convenience and the desire to know results quickly, as well as expectation of commercial profit by the manufacturers of the tests, have been the major stimuli for these developments. Experience has shown that motivated individuals, e.g. diabetic patients, fre- quently perform the tests as well as highly qualified professionals.

The immediate availability of results at the point of care can enable the appropriate treatment to be instituted quickly and patients’ fears can be allayed. However, it is important to ensure that the limitations of any test and the sig- nificance of the results are appreciated by the tester to avoid inappropriate intervention or unnecessary anxiety.

Outside the laboratory

Table 4.1 shows what can be commonly measured in a blood sample outside the normal laboratory setting. The most common blood test outside the labora- tory is the determination of glucose concentration, in a finger stab sample, at home or in the clinic. Diabetic patients who need to monitor their blood glucose on a regular basis can do so at home or at work using one of many commercially available pocket-sized instruments.

Figure 4.1 shows a portable bench analyser. These analysers may be used

Fig 4.1 A portable bench analyser.

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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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