Laboratory Diagnosis of Mycotic Infection

Laboratory diagnosis of mycotic infections

Introduction

A large number of fungal species that are found in the environment have been recognized as important causes of human diseases, mainly in the immunocompromised patient. The modern clinical laboratory, therefore, needs to provide appropriate methods for isolating and identifying the common causes of mycotic infection which leads to a disease condition. The major factors responsible for causing increases in a number of fungal infection have been due to an alteration of the host, particularly in the immune system. This alternation may lead to infection by a microorganism that is normally nonpathogenic or may be part of patient's normal flora. For identification of mycotic disease both clinical and histopathologic are only applied means by which saprophytic fungi can be proven to hold true pathogens.

Early and correct diagnosis of fungal infections is essential for many reasons, including a timely institution of antifungal medication and to decrease the unnecessary use of toxic antifungal agents. Fungi seen in the clinical laboratory are differentiated into the two groups on the basis of appearance of morphology of colony:

1. Yeast produces moist, creamy, opaque, or pasty colonies on media.
2. Mold or Filamentous fungi produce cottony, fluffy, wooly, or powdery colonies.
3. Whereas, an appearance of both yeast like and filamentous form represent dimorphic fungi.

Additionally, some of the medically important fungi like Candida albicans represent germ tube, Cryptococcus neoformans gives rapid urease test positive, Histoplasma capsulatum performs histoplasmin skin test etc. which is identified by laboratory diagnosis of such fungi.

Laboratory diagnosis of fungal infection entirely depends on selection and collection of appropriate clinical sample for culture. Since many clinically fungal infection is similar to the micro bacterial infection, the same specimen is collected and culture for both fungi and mycobacteria. The proper collection and specimen and rapid transport to the clinical laboratory are of major importance for recovery of fungi from the infective sample and helps in proper diagnosis.

Measures performed for the lab diagnosis of fungal infection

There are multiple measures adopted for diagnosis of mycotic infection in the lab, for identification of infective fungi. They are as follows:

1. Macroscopic examination
2. Direct microscopic examination
3. Laboratory culture
4. Biochemical tests
5. Serological examination
6. Histopathological studies
7. Molecular technique

Macroscopic examination

This technique follows the identification of the infective form of fungus without visualizing under a microscope. The sample collected from skin scrapping or hair plucking can be directly viewed under UV light in dark room. For example:

• The dermatophytes such as Microsporum fluoresces when placed under UV light in a dark room, it produces green fluorescence of infective hair or skin.
• Similarly, Malasseqia furfur, which infects the skin, also fluoresces under UV light exposure.

Microscopic examination

Direct microscopic examination of collected clinical sample has been preferred for many years; however, its use should be not for all the case. The mission of clinical microbiology laboratory is to provide rapid and accurate diagnosis, which is done by direct microscopic examination primarily gram staining technique of the clinical specimen submitted for culture. Different microscopic techniques performed are:

KOH mount

In the KOH mount, usually 10% (w/v) KOH is used as the mounting medium particularly for hard specimens like hair and nail. The alkaline solution digests keratin but has no effect on fungi. An alkali- glycerin solution can also be used for such specimen.

Staining technique

Before visualizing under the microscope, the clinical specimen is stained by a different technique for identifying the causative agent. The techniques implied are:

• Gram staining

Gram staining technique is specially used for detection of yeast cells. Yeast cell results in the gram-positive reaction. For example, Candida albicans which are observed in vaginal discharge results in the gram-positive test.

• India ink preparation

It is a negative staining technique where the organism appears as clear disc against a dark (black) background. India ink preparation is done for detection of the capsule containing a fungus which is the extracellular polysaccharide. The encapsulated yeast cells are mostly detected against the black background. It is also used in the identification of Cryptococcus neoformans which is also present in CSF sample.

• Calcofluor white stain

It is a fluorochrome which can be used to rapidly detect yeast cells, pseudohyphae and hyphae in the specimen when examined by fluorescence microscopy. A fluorescence microscope is necessary for identifying fungal cells prepared with calcofluor White. Yeast cells, pseudohyphae, and hyphae display a chalk-white or brilliant apple-green fluorescence with the application of calcofluor white stain.

• Giemsa stain

Gram stain is usually a poor stain to use when examining a specimen for a fungus. Gram stain may be used for examining smears of Candida, Malassezia, and Sporothrix but should not be relied upon to demonstrate the yeast of the other dimorphic fungi.

Laboratory culture

Laboratory culture is done when it is not possible to diagnosis a serious fungal infection microscopically . Is is also preferred to species identification which needs to be established or confirmed . The commonly used media for the culture of fungi are:

Sabouraud dextrose agar-It is commonly used for opportunistic and pathogenic fungi.

Sabouraud dextrose agar with antibiotics-It is preferred for specimen where bacterial contamination is suspected.

Chlamydospore agar/corn meal agar-It is preferred for chlamydospore formation for Candida.

Brain heart infusion agar without antibiotics-For fastidious organism

Brain heart infusion agar with antibiotics-Used for yeast phase, the growth of dimorphic fungi.

Potato Dextrose Agar-In PDA, usually two culture with the specimens are done and the plates are incubated one at 25°C and other at 37°C kept for more than one week. Identification can be done by studying the cultural characteristics like morphology of the culture color and structure of the growth. Similarly, the morphology of conidia and spores produced as well as hyphae quickly the fungus grows and at what temperature the fungus grow.Example Trichophyton rubrum produces wine red diffusible pigment.

Biochemical tests

Although fungi give very few biochemical tests, some of the important biochemical tests are as follows:

1. Urease test
2. Phenol-oxidase enzyme
3. Nitrate reduction test
4. Germ-tube test
5. Temperature-tolerance test

Urease test

The rapid urease test is a most useful biochemical test for screening of urease producing yeasts recovered from respiratory secretions and other clinical specimens. Cryptococcus neoformans gives positive urease test which is a causative agent of fungal meningitis.Also, some candida is also urease positive.

Phenol -oxidase enzyme

Phenol-oxidase enzyme is found in Cryptococcus neoformans is responsible for melanin production .The produced melanin production can decrease lymphocyte proliferation and TNF production, hence it helps in making the organism resistant to lymphocyte attack. Some C. neoformans, however, may not produce phenol oxidase enzyme.

Nitrate reduction test

Besides, rapid urease test, nitrate reduction test and detection of phenol-oxidase production are also useful in identifying Cryptococcus neoformans.

Germ-tube test

Germ-tube test is very significant in identifying Candida albicans as germ tube test is given by C. albicans but not by other Candida species. In this test, a loopful of yeast colony from a culture plate is inoculated into 0.5 ml of human serum in a small test tube and the tube is incubated at 37°C for 2-3 hours. After incubation, the preparation is examined under a microscope for the observation of germ tube. Germ tube appears at hyphal-like an extension from the yeast cell that appears without a constriction at the point of origin from the yeast cell.

Temperature -tolerance test

Cryptococcus neoformans grows at 37°C whereas saprophytic fungi fail to grow even at 31°C.

Histopathological studies

These are useful in pulmonary and other tissue infections. The invasion of tissue or organs by fungi produces a variety of inflammatory reactions. Example Blastomyces dermatitis produces an acute suppurative (pus-forming ) inflammation. Similarly, Aspergillus fumigatus produces necrotizing inflammation (tissue rupture). Hyphae and yeast forms may be also seen in the tissue preparation.

Serological tests

For systemic infection, acute and convalescence pair can be used fungal antigens from blood, CSF can also be detected by the use of monoclonal antibodies, latex agglutination, precipitation, ELISA , etc can also be used in case of systemic infections for the diagnosis of fungal antigens.

Molecular techniques

These techniques are more sophisticated and require high standard instrumentations and expert technicians. Nucleic acid techniques such as hybridization using a specific probe, Polymerase Chain Reaction (PCR) for DNA amplification for the fungal pathogens are the molecular techniques used for diagnosis of selected fungal pathogen.

Lab diagnosis of CSF specimen in fungal meningitis

Most probable organism

Cryptococcus neoformans

Collection of specimen

Lumbar puncture should be done in the same way as for bacteriological sampling, taking precautions to minimize contamination by yeast colonizing the surface of the skin.

Adult- L3-L4

Children-L4-L5

A large volume should be taken if this is clinically permitted taking account of the danger to the patient that is a minimum of 5ml is recommended especially where investigations for cryptococcus in non-AIDS patients are requested.

Transportation

Immediately CSF sample should be taken to the lab without any delay. Refrigeration is not preferred.

Lab diagnosis

A. Direct examination

The specimen must be firstly concentrated by centrifugation before any attempt to carry out the detection of an organism. In direct examination method, after centrifugation of the CSF, the supernatant can be used for direct cryptococcal antigen detection which should be carried out according to the manufactures instructions but with the added precautions of diluting the samples in order to overcome any prozone effect.

Soluble antigen derived from the capsular material can be detected in CSF using latex particle or Staphylococcal cell coated with anticryptococcal antibodies (COAG test). Up to 90% of patients with Cryptococcal meningitis give a positive CSF antigen result.

B. Direct Microscopy

India ink preparation

For India ink preparation, a drop of resuspended sediment is mixed with a similar volume of India ink and visualized under the microscope. Capsules appears as large clear areas surrounding the yeast cells. Later present, capsules are presumptive evidence of Cryptococcus neoformans.

Giemsa stain

Giemsa stain preparation of centrifuged deposit of Cryptococcus may show the capsule surrounding the Cryptococcus neoformans as clear unstained area.It can be used as an alternative to India ink preparation

.C. Lab culture (Isolation of yeast)

A representative loopful of centrifuged deposit should be taken to inoculate the fungal agar media in the usual fashion. All pathogenic yeast grow at 37°C but it is suggested to incubate Cryptococcus neoformans at 28°C.

On sabouraud agar, Cryptococcus neoformans produces moist white cream colored mucoid colonies usually after 2-3 days of incubation. When examined microscopically, the yeast is capsulated but the capsules are often smaller than when seen in the specimen. Occasionally capsules are absent.

D. Biochemical tests

Cryptococcus can be reliably differentiated from Torulopsis by its ability to produce urease. For this, a slant of Christenson's urea agar can be inoculated and incubated at 30°C for 48hours. Conversion of phenolphthalein indicator from yellow to pink or red denotes the alkaline change when urease liberates ammonia from urea.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. 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.