Fungal Infections - Specimen Collection, Transport and Processing

Introduction

Laboratory diagnosis of fungal infection is highly dependent on the quality of specimens. Hence, appropriate collection, transport, and processing of specimens is important for the diagnosis of specimens.

Clinical specimens

Clinical specimens collected for the diagnosis of mycosis include:

1. Respiratory tract secretions

2. Cerebral spinal fluid

3. Blood

4. Tissue

5. Body fluids

6. Bone marrow

7. Urine

8. Vaginal secretions

9. Skin

10. Hair

11. Nail

1. Respiratory tract secretions:

The respiratory tract secretions include sputum, tracheal secretions, and bronchoalveolar lavage (BAL) fluid.

• Sputum samples should be collected in the early morning

• In case of non-productive cough, sputum is induced and then collected

Processing of respiratory tract secretions

• To the sputum sample, an equal amount of N-acetyl-L-cysteine (NALC) is added

• The mixture is vortexed for 10 seconds

• To the mixture, 0.015 M phosphate buffer with pH 7 is added and brought to a 50-volume

• Approximately 0.005 µg/ml chloramphenicol is then added

• The sample is then inoculated into culture media and incubated

2. Cerebral spinal fluid (CSF):

Cerebral spinal fluid (CSF) samples collected for the laboratory diagnosis of fungal infections must be immediately processed. In cases where CSF cannot be immediately processed, the samples must be stored at room temperature (25°C) or incubated at 30°C.

Processing of Cerebral spinal fluid (CSF):

• Cerebral spinal fluid (CSF) is centrifuged

• Sediment is collected for further processing

• Drops of sediment are placed on several areas on the culture agar surface and incubated

3. Blood:

Blood culture is one of the most accurate methods for the diagnosis of fungal infections. Numerous systems are available for the optimal recovery of fungi from samples including:

• BACTEC

• BACT/ALERT & ESP system

• Lysis centrifugation system (for dimorphic fungi such as Histoplasma capsulatum)

Processing for blood culture:

• The blood specimen collected is added to a sterile liquid culture media

• The inoculated media is incubated at an optimum temperature of 30°C for 21 days

4. Tissue, Bone Marrow, Body Fluids

• Tissue samples are minced or ground and 1 ml of the specimen is spread on the surface of the media agar plate. The inoculated media is incubated at 30°C for 4 weeks.

• Bone marrow specimen is directly inoculated on the media plate and incubated at 30°C for 4 weeks.

• The body fluid sample is concentrated by centrifugation. The sedimentation is cultured.

5. Skin, Hair, Nail:

Samples such as skin scrapings, hair cuttings, and nail cuttings are collected for laboratory diagnosis of dermatophytic infections.

The sites from which samples need to be collected are cleaned with 70% v/v ethanol. Samples collected should be placed in a sterile petri plate or paper envelope.

Processing of skin, hair, and Nail

• Skin scales/scrappings are obtained by scraping the surface of the margin of the lesion using a sterile blunt scalpel

• Parts of crusts are removed with the help of sterile scissors

• Infected nail pieces are snipped using sterile scissors, placed into a tube containing a few drops of KoH solution, heated, and then left overnight

• Infected hair is plucked with forceps

• The samples (or wet preparation) are placed on the surface of culture media and incubated at 30°C for 4 weeks

6. Vaginal secretions:

Vaginal secretions are collected for the diagnosis of vaginal candidiasis caused by Candida spp.

7. Urine:

Urine samples collected for laboratory diagnosis of fungal infections are processed as early as possible. In case of delay, the specimen can be kept at 4°C for up to 12 hours.

Processing of urine

• Urine samples are centrifuged

• Sediment is inoculated into culture media supplemented with antibiotics

• The inoculated plate is incubated at 25-30°C for 30 days