Yeasts Identifications

Blastoschizomyces
Candida
Cryptococcus
Loboaloboa
Malassezia
Rhodotorula
Saccharomyces
Trichosporon
Identification.
Yeast-like fungi may be basidiomycetes, such as Cryptococcus neoformans or ascomycetes such as Candida albicans.
1. Ensure that you start with a pure culture; streak for single colony isolation if necessary.
2. Germ Tube Test: lightly inoculated 5 ml of serum, containing 0.5% glucose and incubated at 35oC for 2-3 hours.
Positive = Candida albicans or Candida dubliniensis.
Negative or from HIV positive patient = perform assimilation tests.
3. For the identification of germ tube negative yeasts, morphological (Dalmau plate culture), physiological and biochemical tests are essential.
Dalmau Plate Culture: To set up a yeast morphology plate, dip a flamed sterilized straight wire into a light inoculum (sterile distilled water suspension) and then lightly scratch the wire into the surface of a cornmeal/tween 80, rice/tween 80 or yeast morphology agar plate, then place a flamed coverslip onto the agar surface covering the scratches. Dalmau morphology plates are examined in situ using the lower power of a microscope for the presence of pseudohyphae which may take up to 4-5 days at 26oC to develop. C. albicans also produces characteristic large, round, terminal, thick-walled vesicles (often called chlamydoconidia). The key features to remember are to use a light inoculum and to scratch the surface of the agar with the wire when inoculating.
Physiological and biochemical tests: including fermentation and assimilation studies should be performed. Reliable commercially available yeast identification kits are the API 20C AUX, ATB 32C, MicroScan and Vitek systems
Identification of Common Dermatophytes.
Microscopic morphology of the micro and/or macroconidia is the most reliable identification character, but you need a good slide preparation and you may need to stimulate sporulation in some strains. Culture characteristics such as surface texture, topography and pigmentation are variable and are therefore the least reliable criteria for identification. Clinical information such as the site, appearance of the lesion, geographic location, travel history, animal contacts and race is also important, especially in identifying rare non-sporulation species like M. audouini, T. concentricum and T schoenleinii etc. Note: mating experiments are not practical for the clinical mycology laboratory.
Three genera are recognized:
Epidermophyton:
Smooth thin-walled Macroconidia only present, no microconidia, colonies a green-brown to khaki colour.
Microsporum:
Macroconidia with rough walls present, microconidia may also be present.
Trichophyton:
Microconidia present, smooth-walled macroconidia may or may not be present.
Lactophenol Cotton Blue.
For the staining and microscopic identification of fungi

Method
This stain is prepared over two days.
1. On the first day, dissolve the Cotton Blue in the distilled water. Leave overnight to eliminate insoluble dye.
2. On the second day, wearing gloves add the phenol crystals to the lactic acid in a glass beaker. place on magnetic stirrer until the phenol is dissolved.
3. Add the glycerol.
4. Filter the Cotton Blue and distilled water solution into the phenol/glycerol/lactic acid solution. Mix and store at room temperature.
Sabouraud's Dextrose Agar for Dermatophytes
Sabouraud's Dextrose Agar with Cycloheximide, Chloramphenicol, Gentamicin and Yeast Extract.
For the primary isolation and cultivation of dermatophytes.

Method:
1. Soak all ingredients, except Gentamicin, in 100 ml water.
2. Boil remaining water, add to soaking ingredients, and bring to boil to dissolve, stirring well to prevent from charring.
3. Add the Gentamicin. Mix well.
4. Dispense for slopes if required.
5. Autoclave at 121C for 10 minutes. Remove and slope, or pour for plates as required.

Sampling for FUNGAL Infections and Culture

Skin Scrapings and Swabs
In patients with suspected tinea or ringworm any ointments or other local applications present should first be removed with an alcohol wipe. Using a blunt scalpel, tweezers, or a bone curette, firmly scrape the lesion, particularly at the advancing border. A bone curette is safe and useful for collecting specimens from babies, young children and awkward sites such as interdigital spaces. If multiple lesions are present choose the most recent for scrapings as old loose scale is often not satisfactory. Any small vellus hairs when present within the lesions should be epilated. The tops of any fresh vesicles should be removed as the fungus is often plentiful in the roof of the vesicle.
In patients with suspected candidiasis the young "satellite" lesions which have not undergone exfoliation are more likely to yield positive results if they are present. Otherwise the advancing scaly border should be scraped. When lesions in the flexures are moist and very inflamed it is more satisfactory and less painful to roll a moistened swab firmly over the surface.
In patients with suspected cutaneous manifestations of systemic pathogens scrap the lesions with a bone curette or blunt scalpel as for tinea. A biopsy may be required in some cases.
NOTE: Following the collection of skin scales all scraped lesions should be firmly rubbed with a swab moistened in BHI broth.
Skin Scrapings, nail scrapings and epilated hairs where tinea is the provisional diagnosis:
1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimen onto two slopes containing cycloheximide (actidione) i.e. one DERMASEL agar slope and one LACTRITMEL agar slope also containing chloramphenicol, gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.
Skin scrapings and swabs where candidiasis is the provisional diagnosis:
A. Skin scrapings:
1. Make a wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar slopes containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.
B. Skin Swabs:
1. Smear swab onto heat sterilized glass slide for Gram stain.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 35C. Maintain cultures for 4 weeks.
3. Where secondary bacterial infection is suspected, and separate swabs for routine bacteriology were not collected, the swab should first be inoculated onto a blood agar plate, followed by the Sabouraud's agar containing the antibiotics and then placed into Brain Heart Infusion Broth. All cultures should be incubated at 35C. Maintain cultures for 4 weeks.
NOTE: Where a dermatophyte is suspected or to be excluded a Sabouraud's agar slope containing cycloheximide and incubated at 26C may be included.
Scrapings from the groin, feet or nails where either a dermatophyte or Candida species may be isolated. This includes the possibility of a non-dermatophyte onychomycosis.
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar containing chloramphenicol and gentamicin, but NO cycloheximide (as for Candida) and incubate at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto a DERMASEL agar slope containing cycloheximide (actidione), chloramphenicol and gentamicin and incubate cultures at 26C. Maintain cultures for 4 weeks.
4. Where a moistened swab has also been collected from the same site as the scraping, inoculate this onto a Sabouraud's dextrose agar slope containing chloramphenicol and gentamicin, but NO cycloheximide and incubate at 26C. Maintain cultures for 4 weeks.
Skin scrapings from patients with suspected pityriasis versicolor:
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy along with the cellotape stripping taken at the time of collection.
2. Inoculate scrapings onto an DIXON'S agar slope for isolation of Malassezia furfur and incubate cultures at 26C. Maintain cultures for 4 weeks.
3. Inoculate specimen onto Sabouraud's dextrose agar with chloramphenicol and gentainicin but NO cycloheximide (actidione) and incubate cultures at 26C. To exclude other yeasts like Candida. Maintain cultures for 4 weeks.
4. If dermatophytes are to be excluded also inoculate onto DERMASEL agar slope and incubate cultures at 26C. Maintain cultures for 4 weeks.
Skin scrapings from patients where a systemic pathogen is suspected:
1. Direct Microscopy: Wet mount preparation in KOH for direct microscopy. Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto:
(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin but NO cycloheximide (actidione) and incubate duplicate cultures at 26C and 35C; and
(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Sputum, Bronchial Washings and Throat Swabs
Many opportunistic mycoses have a pulmonary origin following the inhalation of fungal propagules. Bronchial washings and sputa should be collected upon rising in the morning as overnight incubation and growth of fungi in the lungs will increase the likelihood of isolating pathogenic fungi. Patients should not eat before specimen collection. Twenty-four hour samples are unacceptable because they become overgrown with bacteria and fungal contaminants. It should also be stressed that bronchial washings and sputa will usually be contaminated with throat flora. For this reason interpretation of results may be difficult from poor quality specimens.
Throat specimens are obtained by rolling a moist sterile swab over the affected area. However, if Candida is suspected the affected area will need to be scraped with a sterile tongue depressor.
All specimens must be sent to the laboratory and processed as soon as possible, a delay of longer than two hours at room temperature may impede the detection of some fungi. Store at 4C if short delays in processing are anticipated.
Unless it is already sufficiently fluid to pipette with a Pasteur pipette, sputa may need to be emulsified by shaking with l2-20 sterile glass beads and about 3-5ml of sterile distilled water, depending on the volume of the original specimen. Any bits of blood, pus or necrotic material should be plated directly onto media.
(1) Make wet mount preparations in KOH (l drop) and Gram stained smears (l drop) of all suspicious areas. The PAS stain may be necessary if the KOH preparation is unsatisfactory.
(2) Inoculate sample onto:
(a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C; and
(b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Blood and Bone Marrow
The laboratory should be informed by the physician if fungal septicemia is suspected because special media are necessary for the optimum recovery of fungi. Numerous blood culture systems are available; however all systems must be vented to atmospheric air and incubated at 30C to maximize the rate and time of recovery of fungal organisms.
Aseptically collect 10 ml of blood and prepare several smears for Giemsa, Gram and PAS staining. Culture the remaining specimen by one or more of the following methods. With bone marrow aspirates the initial material is generally used for making smears for Giemsa staining, the remaining 3-5 ml of marrow and blood may be cultured on the media listed below.
1. Direct culture method: Inoculate 0.5-1.0 ml of buffy coat, prepared by centrifuging 5-10 ml of blood, onto the surface of the media listed below. This inoculum can then be spread over the surface of the agar with a sterile inoculating loop and the plate incubated aerobically at 30C. This method is suitable for small low volume laboratories where there are few requests for fungal blood cultures. Cultures should be maintained for 4 weeks.
2. Biphasic culture bottle: The recovery of fungi from blood may be enhanced by using a biphasic bottle containing a slant of brain heart infusion agar and 60-100 ml of BHI broth. A ratio of 1:10 to 1:20 (blood to broth) is recommended, a minimum of 5.0 ml of blood is required for each culture bottle. The biphasic culture bottle is kept vented and is tilted daily to allow broth to flow over the agar surface. These cultures must be carefully checked daily for growth. Because fungi will not turn the broth very cloudy it is imperative to frequently Gram stain the bottle contents to detect fungal elements. Cultures should be incubated at 30C and maintained for 4 weeks.
3. Membrane filter technique: This is a superior technique to the vented biphasic blood bottle used to concentrate and culture specimens of blood and CSF. Briefly, specimens are treated sequentially with Triton-X and sodium carbonate solutions to lyse blood cells and then filtered by vacuum through a 0.45 um membrane. This membrane is then placed onto the media listed below.
4. Lysis centrifugation isolator system: The Wampole Isolator system has been found to significantly improve the recovery of fungi from blood and is strongly recommend by Koneman and Roberts (1985) as the method of choice for processing blood cultures from patients with suspected fungal septicemia. The Isolator utilizes a tube that contains components that lyse leukocytes and erythrocytes and also inactivate plasma complement and certain antibiotics. Once lysed, the cells release the microorganisms contained within them, and the centrifugation step in the procedure serves to concentrate the organisms in the blood sample. This concentrate is then inoculated onto the surface of appropriate culture media listed below. Ten milliliters of blood are required for each tube and cultures should be incubated at 30oC and maintained for 4 weeks.
5. Bactec: Bactec have produced a special fungal media (BACTEC Fungal Medium) for enhanced fungal blood culture using their non-radiometric (NR) instruments. Once again, blood cells are lysed by the medium to enhance recovery of fungi. Note antimicrobials have also been added to limit the growth of bacteria.
Primary isolation media for blood and bone marrow culture: (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26oC and 35oC; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for 4 weeks.
Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26C for 4 weeks to exclude the presence of a slow growing fungus
Urine should be collected first thing in the morning after overnight incubation in the bladder. A midstream clean catch or catheterized specimen is best, as this minimizes the presence of genital flora. Do not use urine from a collection bag or bed pan. Twenty-four hour urine samples are unacceptable.
Yeasts recovered from routine urine bacteriology cultures of catheterized urine or urine obtained by sterile procedure should be identified and reported regardless of colony count. However, the isolation of yeasts from clean catch specimens must be interpreted with caution and is not significant without additional support from other clinical and laboratory investigations.
Note: Negative bacteriological cultures from patients with clinical evidence of an infection should be sealed with tape and maintained at 26C for 4 weeks to exclude the presence of a slow growing fungus.
Urine samples must be processed as soon as possible, a delay of longer than two hours at room temperature may impede the detection of some fungi. Store at 4oC if short delays in processing are anticipated.
(1) Centrifuge the urine for 10-15 minutes at 2000 rpm. Decant the supernatant and pool the sediment if necessary.
(2) Prepare a direct smear of the sediment in KOH for direct microscopy. Note PAS, Gram or India ink preparations may also be helpful.
(3) Inoculate 0.05-0.1 ml of the sediment onto Sabouraud's agar with gentamicin and chloramphenicol and incubate duplicate cultures at 26C & 35C. Maintain cultures for 4 weeks.
Cerebrospinal Fluid (CSF):
Three to five milliliters of CFS is optimal for fungal investigation, however lesser volumes are often received and should be processed. CSF specimens should be transported to the laboratory as soon as possible and processed promptly. If there is a delay do not refrigerate the samples, rather they should be left at room temperature or incubated at 30C. The specimen should be centrifuged. Keep the supernatant for cryptococcal antigen testing and process the sediment as follows.
(1) For direct microscopy use 1 drop of the sediment to make an India ink mount.
(2) Resuspend the remaining sediment in 1-2 ml of CSF and inoculate onto; (a) Sabouraud's dextrose agar with chloramphenicol and gentamicin and incubate duplicate cultures at 26C and 35C; and (b) Brain heart infusion agar (BHIA) supplemented with 5% sheep blood and incubate at 35C. Maintain cultures for at least 4 weeks.
Note: Cultures from patients undergoing treatment for cryptococcal meningitis should be maintained for 3 months, so that dormant viable cells, which do not start to grow until after a one month period, will not be missed.

Entero-invasive E.coli (EIEC).

Entero-invasive E.coli (EIEC).
Enteroinvasive strains of E.coli (EIEC) are strains produce dysentry with direct penetration, invasion, and destruction of the intestinal mucosa. This diarrheal illnesses is very similar to that produced by Shigella. The EIEC infections seem to occur in adults and children alike (Mahon and Manuselis 2000).

EIEC strains are generally lysine decarboxylase negative, non motile, and lactose negative (Brenner et al., 1973). EIEC apparently lack fimbial adhesions but do not possess a specific adhesions that as in Shigella, is thought to be an outer membrane protein. Also, like Shigella, EIEC are invasive organisms. They do not produce LT or ST and, unlike Shigella, they do not produce the Shiga-toxin (Hale et al., 1997).

1.3.11.1. Pathogenesis.
The pathogenesity is resemble to Shigella pathogenesis. Both organisms have shown to invade the colonic epithilium, a phenotype mediated by both plasmid and chromosomal loci. In addition, both EIEC and Shigella Spp elaborate one or more secretory enterotoxins that may play roles in diarrheal pathogenesis (Nataro, 2001).

1.3.11.2. Invasiveness.
The current model of Shigella and EIEC pathogenesis comprises (i) Epithelial cell penetration, (ii) Lysis, of the endocytic vacuole, (iii) intracellualr multiplication, (iv) directional movement through the cytoplasm, and (v) extension into adjacent epithelial cells (Nataro, 2001).

EIEC and Shigella both cause disease by invading intestinal epithelial. Infection is by ingestion, only a small number of bacteria need to be swallowed as they are relatively resistant to gastiric acid and bile and pass readily into the large intestine where they multiply in the gut lumen. The bacteria passes through the overlying mucus layer, attach to the intestinal epithelial cells and are carried into the cell by endocytosis into the endocytic vacuole which then lysis.

The ability to cause the vacuole to lyse is an important virulence attribute, as organisms unable to do this can’t spread to neighbouring cells. After lysis of the vacuole the bacteria multiply within the epithelial cell and kill it. Spread to neighbouring cells leads to tissue destruction and inflammation which cause dysentry. Pathogenicity in Shigellae and EIEC depends on chromosomal and plasmid genes (Green Wood et al., 2002).

1.3.11.3. Epidemiology.
Epidemiology and ecology of EIEC have been poorly studied. Documented EIEC out breaks are usually foodborne or water borne, although person to person transimission does occur. Infections are usually foodborne but there is also evidence of cross infection. The most common serogroup is O124 (Green Wood et al., 2002).