Sample Collection and Laboratory Diagnosis

Introduction

Fungi

Fungi are the microorganism which is both multicellular and unicellular. They are a saprophytic as well as a eukaryotic organism. Fungi are larger than bacteria, an individual cell diameter of fungi ranges from 1-30 µm. They exist in three different form: simplest form unicellular, multicellular, and dimorphic. Thus, morphologically fungi can be divided into 3 groups. They are;

1. Unicellular - Yeast
2. Multicellular - Molds
3. Dimorphic fungi - yeast or filamentous form

Fungi can be aerobic or facultative anaerobes; in a case of mold, it is an aerobic organism and in the case of yeast, it is facultative anaerobes. Fungi are composed of a wide arrangement of organisms that are unique. In spite of this morphological variation in fungi, most or all of the fungi share following same characteristics;

• The cell wall of fungi is made up of chitin.
• The cell membrane of fungi has a presence of ergosterol.
• They are non-photosynthetic so shows a heteromorphic mode of nutrition.
• Reproduction can be by both: sexually or asexually, by means of spores.
• Storage form of carbohydrate is glycogen.
• Fungi lack susceptibility to antibacterial agents.

Distribution

Fungi are found everywhere commonly in soil, air and water. They are called saprobes organism because most of this organism live a saprophytic existence in nature. Chlorophyll is lacking in them. Fungi and molds are found in shady and moist places whereas, yeast is found in moist place only. Similarly, fungi are also found as normal flora of human body. The most common fungi, which is normal flora and found in an oral cavity, genital tract, and gastrointestinal tract is Candida species.

Mycotic infection

Fungi have the capacity to cause infection in humans. There are a number of species of fungi which are responsible for causing different diseases in human. More than 50,000 species of fungi exist in nature but only 100-150 species are generally meant to cause human disease and approximately 25 species are responsible for causing the majority of the human diseases. Thus, the diseases caused by fungi are called mycoses or the infection of fungus on a body is known as mycotic infection. Fungi can cause disease in different anatomical position in our body. Fungal infection is not generally communicable from the usual sense from person to person or animal to person but dermatophytes which are a particular group of fungi that infect only superficial keratinized structures (skin, hair, nail) shows the transmission. During accidental injury, a human may become the host for fungi by inhaling spores or by an introduction of fungal elements into the tissue by trauma leading to fungal infection. Mycotic infection may lead from superficial exudates to serious or life-threatening diseases. Major infections caused by fungi are;

• Superficial mycosis - e.g.: Athlete’s foot
• Subcutaneous mycosis – e.g.: Sporotrichosis
• Systemic mycosis – e.g.: Histoplasmosis

The sample for laboratory diagnosis of a fungal specimen is taken from various infective sites with different methods of collection. The sample then collected should be employed to diagnosis the fungal infection. The fungal microorganism most commonly found in various specimens is listed below:

Sample collection technique and transportation

Laboratory diagnosis of mycotic infection begins with appropriate specimen collection and transportation. For the identification of fungal infection, primarily it is concerned in the visualization of colony morphology and study of microscopic features. The sample collection should be done carefully so that proper infection can be identified and diagnosis becomes easy. Different biochemical tests may be required to differentiate between the genes and species of mycosis causative agent as well as a serological test is performed for laboratory diagnosis. But medically important fungi generally not being identified by serological and biochemical tests also the stage of infection cannot be confirmed rather confirmed by microscopic view of some unique structure.Laboratory technique is implied for identification of fungal infection and diagnosis. Methods for collection of different sample from infective site for laboratory diagnosis of fungal diseases are mentioned below:

1. Skin scrapping

The skin lesion is taken mainly for identification of fungal infection by laboratory diagnosis. It is preferred in a case of superficial infection that is mainly confined to the skin and other external structures like hairs, nails, and some other part. Microorganism responsible for superficial infection is Epidermophytonspecies, Microsporum species, Trichophyton species, etc.

Procedure

1. At very first, the affected area should be cleaned with 70% ethanol.
2. By the use of a sterile and blunt scalpel, an active portion of a lesion is obtained from the surface of the infected skin.
3. The superficial material should not be sampled rather peripheries of the skin lesion is preferred.
4. Skin crapping on scalp:
   The sample should include hair stubs, the content of plugged follicles and skin scales.
5. Then the scrapping is placed in a clean envelope or on a glass slide which is then pressed with another glass slide. The color of the paper over which slide are kept should be dark so that the specimen is easily visible.
6. Finally, the collected specimen should be labeled with the patient information and then transported to the laboratory for diagnosis purpose.

2. Nail clipping

Keratinized portion nail, is also a possible site for residing of fungus, should be sampled as nail clipping. Nail clipping is taken from any of the discolored or thickened or critic part of the nail. Following steps should be considered during specimen collection:

Procedure

1. Firstly, Nail with polished should be removed.
2. A Nail is then wiped with 70% alcohol.
3. Debris under the nail is collected which is then placed in a clear glass tube.
4. Nail pieces are collected by taking the infected part of the nail with the help of scissors.
5. The outer surface of the nail is scrapped and discarded.
6. The scrapings from the diseased area of the nail are taken and placed with previous debris collected from under the nail.
7. Finally, the collected specimen should be labeled with the patient information and then transported to the laboratory for diagnosis purpose.

3. Sputum

Fungal infection can be determined from sputum sample of a human. Microbiologist instructs the patient in a difference between sputum and spit. The patient is given a clean, dry, wide-necked, leak -proof, a screw-cap container to produce and collects deep coughed sputum. There is different precaution taken under consideration during collection of the sputum sample.

Precaution

• A patient should brush teeth and mouth should be rinsed thoroughly before sample collection.
• The container used to collect sputum should be clean, sterile and dry.
• The only sputum should be collected but not saliva, mucus, or nasal secretion.

Procedure

1. Generally, the first early morning sputum sample is preferred.
2. The patient should brush teeth and rinse mouth well.
3. The sputum is raised by coughing and collected in a sterile container.
4. The sample collected is then centrifuged after liquefaction and sedimentation are used for inoculation as well as direct microscopic examination.

4. Hair plucking

Sometimes hair plucking is also collected from the infected site of hair. Before collection of the hair sample, removing of dull broken hairs from the margin of the lesion using tweezers is preferred.

Procedure

1. The infected hairs are plucked by using a sterile fine forceps.
2. At least 10-20 affected hairs are removed with forceps.
3. This sample is then placed in a sterile petri dish.
4. While collecting hair plucking, both the hair shaft and hair root should be obtained.

Labelling

• Label the specimen with patients’ information.
• Identify the specimen source clearly.
• Note the suspected diagnosis if possible.
• Ensure that tubes are a shield.

Transportation

• During transportation, refrigeration of the specimen is not done.
• Submission of collected sample at room temperature.

5. Cerebrospinal fluid (CSF)

CSF is the sterile part which is free of normal flora. But whenever infections occur, the presence of a microorganism in CSF sample is detected. The collection of CSF sample is done by an experienced medical officer. It requires very careful and aseptic condition so that entering of microorganism in Central Nervous System (CNS) is prevented.

Site selection

Selection of a site is taken under consideration while collecting the CSF sample. The specimen is collected using strict aseptic technique and by an expert, a sterile needle is inserted between the 4^th and 5^th lumber vertebrae to avoid spinal cord damage.

• Adult = L3 – L4
• Children = L4 – L5

Methods of collection

1. A patient is allowed to be in such a position in which they should arch his/her back so that their head nearly touches the knees.
2. In order to draw sample from lumber puncture i.e. adult: L3-L4 and children: L4-L5, skin along the line of sample drawing is disinfected by
3. Now by an experienced physician, a needle is carefully introduced between the fourth and fifth lumbar vertebra (L4-L5) to avoid spinal cord damage.
4. As soon as CSF sample starts to drop from inserted needle, its position in the subarachnoid space is established.
5. A sterile tube is then placed to hold the sample, normally 5-10ml of CSF sample is recommended.

• After collection of required sample, the specimen is labeled with the patient information i.e. Name Age Date of a collection, etc.
• Labeling also includes an indication of any therapy being given to a
• patient.
• Then immediately deliver the samples with a request form to the laboratory. Any delay may produce cell count that doesn’t reflect the clinical situations to the patients.

Transportation

• Transportation of CSF sample is done as soon as possible.
• CSF sample should be hand delivered to the laboratory.
• The specimen should never be refrigerated.
• If a delay in transportation is to happen due to any reason which disturbs the rapid processing, a specimen should be incubated at 37°Cor left at room temperature but refrigeration is not preferred.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries.Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.