INFECTIOUS DISEASE

PORTUGUESE

MYCOLOGY - CHAPTER ONE 

INTRODUCTION TO MYCOLOGY 

Dr Art DiSalvo
Emeritus  Director, Nevada State Laboratory
Emeritus Director of Laboratories, South Carolina Department of Health and Environmental Control

Dr Errol Reiss Ph.D.
Research Microbiologist (retired)
Centers for Disease Control and Prevention
Atlanta, Georgia, USA

Dr Errol Reiss' contribution to this Section is written in his private capacity. No official support or endorsement by the Centers for Disease Control and Prevention, Department of Health and Human Services is intended nor should be inferred.

 

TURKISH

ALBANIAN

Figure 2
Figure 2: Radiating hyphae of a mycelium
Credit: Dr. A.H.R. Buller, 1931

Figure 3A
Candida albicans growing as a unicellular budding yeast. Growth at 37°C with aeration in yeast-peptone-dextrose broth medium. In this image, unstained cells are magnified x400 (phase- contrast microscopy).

CLASSIFICATION

Fungi defined
Fungi are simple eukaryotes with chitin-containing rigid cell walls and are organized in the Kingdom Fungi. They do not contain chlorophyll and are not plants. Medical mycology is mostly concerned with microfungi, specifically zoopathogenic fungi. They grow in two forms:

Mold
A non-motile thallus constructed of apically elongating walled filaments (hyphae). A web of filaments constitutes a mycelium (Figures 1 and 2)

Yeast (blastoconidia).
A unicellular fungus that reproduces by budding. Small, round projections from the ellipsoid shaped parent cell are produced during mitosis followed by migration of the nucleus and cytoplasm into the bud. Finally, cytokinesis occurs forming a new daughter cell. Buds may be solitary or in chains.

Some yeasts multiply by fission. Events in the yeast cell cycle are finely orchestrated. A visual 3-D representation of the yeast cell cycle can be found here.

Candida albicans is a yeast-like fungus that grows in a variety of forms: yeast, pseudohyphae (a transitional form) and hyphae. Pseudohyphae can give rise to yeast cells by apical or lateral budding. Yeast can also convert to a hyphal form. All three forms are found in tissue invaded by the fungus. Figures 3 A - C show C. albicans growing as a unicellular budding yeast under some environmental conditions and as a filamentous fungus under other conditions.

Figure 4 shows a colony of yeast and of a mold growing in agar plate cultures.

WHERE DO FUNGI GROW?

Most are saprobes that decompose dead organic matter. In contrast to plants and algae, fungi are “heterotrophs”: they cannot make their own food and instead obtain it by uptake of organic matter. Plant pathogenic fungi cause damage to food crops, trees, and other plants. Some fungi are “commensals” living on the mucous membranes and skin of mammalian hosts. Various estimates of the number of fungal species range upwards of 1 million (Heitman, 2011). About 300 species are known human pathogens but any fungus capable of growth at 37 degrees C is potentially pathogenic in a suitably compromised host.
 

Figure 3B 
At higher magnification a budding yeast is seen with a septum formed between the daughter bud and the mother cell. The unstained cell is magnified x1,000 (phase-contrast)

Figure 3C
At 37degrees C a C. albicans yeast cell is shown germinating. (i.e., forming a germ tube). This then grows into a filament (hypha) with a septum between cells. (x1000).

Figure 3 A-C
©  Phillip Stafford
Dartmouth Medical School
Hanover, New Hampshire and The MicrobeLibrary

FUNGI DIFFER FROM BACTERIA IN THEIR ORGANELLES AND METABOLISM

• Cellular organelles

Capsule
Polysaccharide capsules of bacterial pathogens are virulence factors. A major fungal pathogen, Cryptococcus neoformans (also C. gattii). has such a capsule.

Cell walls
Bacterial cell walls contain peptidoglycan, lipopolysaccharide, and teichoic acid. Fungal walls contain glucan, mannan, and chitin.

Cytoplasmic membranes
Membranes of fungi contains ergosterol, not present in bacteria. The synthesis of and binding to ergosterol are potent antifungal drug targets.

Episomes and plasmids
Bacterial resistance to antibiotics is mediated by extrachromosomal DNA; no such mechanisms are known to exist in fungi.

Nucleus
As eukaryotes, fungal genes are organized into chromosomes, enclosed in a nuclear membrane. Baker’s yeast, Saccharomyces, has 16 chromosomes. Bacteria, as prokaryotes, have a single chromosome, not enclosed by a membrane, but packed into part of the cytoplasm, the nucleoid, occupying ~1/3 of the cell volume.

Ribosomes
Bacteria 30s + 50s form 70s ribosomes; fungi 40s + 60s form 80s ribosomes

Dimorphism
Some fungi undergo morphogenesis into two forms, such as yeast and mold forms. This feature is absent in bacteria

• Metabolism
  Bacteria are aerobic or anaerobic; fungi during tissue invasion of humans are aerobic, but metabolism by fungi under anaerobic conditions is known, e.g., fermentation by Saccharomyces beer yeast occurs at low oxygen concentrations. Energy transduction in bacteria occurs at the cell membrane; in fungi mitochondria perform this function.
   
• Reproduction
  Bacteria reproduce by binary fission to two identical daughter cells. Fungi reproduce in various ways: budding, linear extension of the growing tips of hyphae, and by the production of various types of spores, which in fungi are called conidia.
   
• Size
  The volume of a typical bacterium, E. coli , is 1 µm3, diameter 1 µm, and length ~2 µm. See here.

In contrast, A budding yeast cell has a V = 42 µm3 (haploid strain) and V= ~82 µm3 (diploid strain), diameter 3-6 µm. See here.

These differences in cellular organization help explain why antibiotics active against bacteria are, with exceptions, inactive against fungi. On the other hand, similarities between the organization and metabolism of fungal cells and human cells complicates development of antifungal agents with selective toxicity for fungi.
 

CLASSIFICATION
There are two types: Biological classification and classification based on the primary site of pathology. Students of medicine will find the second type of classification most useful.

More Information

Classification of Fungi based on the Primary Site of Pathology

Superficial mycoses
This category is typified by pityriasis versicolor, caused by Malassezia species. This yeast grows on the non-living keratinized outer layer of the skin of humans and dogs also includes dandruff and other forms of seborrheic dermatitis, rarely the cause of invasive disease.

Cutaneous mycoses
Dermatophytosis, also known as ringworm, is caused by Trichophyton and Microsporum species. They are restricted to grow on the non-living keratinized outer layer of skin of humans, dogs, and cats. Medical terminology assigns a name to the diseases according to the body site affected: Tinea capitis is scalp ringworm, tinea cruris is jock itch, tinea unguium fungal nail infections, etc. A completely different category is the cutaneous site of disseminated mycoses. Skin is a frequent site for disseminated blastomycosis.

Opportunistic mycoses

Opportunistic yeasts and Pneumocystis
Candidiasis includes mucocutaneous and deep seated disease caused by C. albicans and non-albicans Candida species yeasts. Their ecologic niche is the skin and mucosae of warm-blooded animals and humans. Cryptococcal meningo-encephalitis is caused by the environmental yeasts Cryptococcus neoformans and C. gattii. Pneumocystis pneumonia (“PCP”) is caused by Pneumocystis jirovecii, an obligate endogenous commensal of the human lung.

Opportunistic mold disease
Disease is encountered in debilitated or immunocompromised hosts. The causative agents are non-pigmented molds that are ubiquitous in the environment and cause disease when their conidia are inhaled by a susceptible host or when the conidia alight on skin of burn patients or on wounds. Invasive pulmonary aspergillosis is caused by Aspergillus fumigatus and related species. Mucormycosis is caused by various Mucorales species especially Rhizopus oryzae (syn: R. arrhizus) including rhinocerebral mucormycosis occurring in diabetic ketoacidosis (figure 5). Fusarium species mycosis includes sino-pulmonary-disseminated disease and, in immune normal persons, keratitis (due to penetrating injury or contaminated contact lenses.) Scedosporium species cause pulmonary disseminated disease and, in immune-normal persons, eumycetoma most often resulting from injury during barefoot labor.

Subcutaneous mycoses of implantation
Subcutaneous mycoses are confined to the subcutaneous tissue and systemic spread is rare. Following a penetrating injury with thorns, splinters these agents can develop into deep, ulcerated skin lesions, subcutaneous cysts, or slowly enlarging warty masses.

Sporotrichosis is the most common human subcutaneous mycosis, worldwide in its distribution, also affecting cats. It is caused by the dimorphic fungus Sporothrix schenckii.

Melanized fungi (formerly referred to as “dematiaceous”) cause a spectrum of disease separated into three categories:

Chromoblastomycosis described as warty, slow growing tumor-like cutaneous-subcutaneous masses caused by Fonsecaea pedrosoi, among other species, with the characteristic dimorphic tissue form of round copper-colored muriform cells.

Phaeohyphomycosis is a term derived from the histopathologic appearance of the fungi in cutaneous-subcutaneous cysts: dark yeast-like, pseudohyphae-like, or variously shaped hyphae or a combination of forms. (”Phaeo” from the Greek=dark.) Exophiala dermatitidis formerly “Wangiella dermatitidis” accounts for approximately 30% of human isolates in the U.S.A. (Zeng et al., 2007.)

Eumycetoma. The hallmark of this mycosis is a triad of tumefaction, swelling, and sinus tracts draining “grains” (masses of fungal hyphae) occurring mostly on the extremities, i.e., “Madura foot”. Causative agents are many including Scedosporium and Madurella species. Exposure follows puncture wounds during barefoot labor in the endemic tropical and subtropical areas of the world.
 

DIMORPHIC FUNGI

Some fungi have two growth forms such as certain soil-dwelling molds that are primary respiratory pathogens. Their conidia become airborne and, when inhaled, can survive and undergo morphogenesis to the pathogenic yeast form at 37 degrees C. Specimens, such as sputum, when plated on mycologic medium and incubated at 30 degrees C, grow as molds. This category includes the commensal yeast, Candida albicans which in tissue invasion may assume conformations of yeast, pseudohyphae and true hyphae. Dimorphism in fungal pathogens includes Coccidioides species, filamentous in the environment, converting to endosporulating spherules in the human or animal host.

Endemic Mycoses caused by Dimorphic Environmental Molds
Several soil-inhabiting saprobic fungi have gained the capacity to parasitize mammals causing systemic infection in immune-normal individuals. Disease occurs in defined geographic areas, following inhalation of conidia, beginning asymptomatically in the lungs and progressing to an influenza-like illness or pneumonia. Once inhaled these agents convert from mycelial to yeast or spherule form in the host. If the inhaled dose of conidia is high and the host immune response is insufficient extrapulmonary dissemination can ensue.
Persistence, dormancy, and reactivation may also occur. The agents are Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides, brasiliensis, P. lutzii, Lacazia loboi, Coccidioides immitis, C. posadasii, Talaromyces (formerly Penicillium) marneffei. Each genus has its own predilection for various organs which will be described in discussing the individual diseases.

Coccidioidomycosis
Coccidioidomycosis has two major endemic areas in the U.S.; the California endemic area centered in the San Joaquin valley and the Arizona endemic area centered in Maricopa County including Phoenix and Pima County including Tucson.

Histoplasmosis
The endemic area for histoplasmosis is along the river valleys of the central U.S., overlapping with the blastomycosis area which extends into Canadian provinces bordering the Great Lakes and northern Ontario.

Paracoccidioidomycosis
This is a rural disease endemic to Mexico, and Central and South America, especially in coffee growers of Colombia, Venezuela, and Brazil. Lobomycosis is encountered in the Amazon Rain Forest ecosystem and is transmitted by traumatic lesion from splinters or bites of insects, snakes, rays.

Sporotrichosis
This is also considered in this group, since it is dimorphic, although its geographic distribution is world-wide, there are highly endemic areas in Brazil, India, Mexico, Japan, Peru, Uruguay and South Africa. Sporothrix schenckii ecologic niche and route of infection are different in that transmission occurs through traumatic implantation from thorny plants, wood splinters, sphagnum moss, and hay.

Talaromycosis
Talaromyces (was Penicillium) marneffei is endemic in SE Asia, especially in Thailand, also in Southern China and Hong Kong. Alone among these endemic mycoses T. marneffei rarely infects immune normal humans.
 

In addition to the yeast and mold growth forms referred to above, intermediate forms exist as “pseudohyphae” in Candida albicans.


MYCOTIC DISEASES

There are four types of mycoses:

• Hypersensitivity. An allergic reaction to molds and their airborne conidia.
• Mycotoxicoses. Poisoning of humans and lower animals by ingestion of food or feed contaminated by low molecular wt fungal toxins produced by pre-harvest infestation or during storage of peanuts, grains (Pitt and Miller 2016).
• Mycetismus. Poisoning after ingestion of certain mushrooms (50-100 cases/year in U.S.A). (Smith and Davis 2016.)
• Infection. Inhalation, ingestion, or implantation of infectious propagules that progresses to disease via tissue invasion, evoking a host immune response. We shall be concerned only with the last type: disease resulting from infection with pathogenic fungi.
  
  Host and Microbial Factors affecting Pathogenicity

Host Risk Factors (Muskett et al., 2011)
Patients receiving immunosuppressive therapy for maintenance of a transplanted organ or stem cell transplant, cancer chemotherapy, autoimmune disease, and in persons living with HIV/AIDS.

• Prolonged ICU stay, mechanical ventilation.
• Very young (< 1 mo.) or aged (>65 y) patients
• Inborn or acquired deficits: chronic granulomatous disease, cystic fibrosis, diabetes.
• Invasive diagnostic and surgical procedures: abdominal surgery, prosthetic implants, indwelling catheters, renal dialysis.
• Travel to or residence in an endemic area.
• Occupational or recreational exposure: Barefoot labor, gardeners exposed to thorny plants, workers in demolition of old buildings.

Microbial Factors
Elucidation of mechanisms of pathogenicity is an active area of research. The following are a sampling of our understanding of factors affecting fungal pathogenesis.

• Adhesins. Adherence to endothelial cells is a prime requisite for tissue invasion. Examples of fungal adhesins are: ALS of C. albicans, BAD1 of Blastomyces dermatitidis.
• Biofilm formation on biomaterials. Ability of fungi to adhere to and embed in biofilms increases their resistance to antifungal agents.
• Capsule. Encapsulated microbes are resistant to phagocytosis and are implicated in CNS disease. Among fungi, Cryptococcus neoformans has an acidic high molecular wt polysaccharide capsule that is antiphagocytic and may facilitate endothelial crossing into the CNS (Zaragoza et al, 2009).
• Melanin. Melanin in fungal cell walls makes them resistant to phagocytosis and killing. Some examples of melanized fungi include Cryptococcus neoformans, Paracoccidioides brasiliensis, Sporothrix schenckii (Nosanchuk et al., 2006).
• Resistance to the oxidative burst of polymorphonuclear neutrophilic granulocytes. Primary respiratory pathogens, e.g.: Blastomyces, Histoplasma, Paracoccidioides, and Sporothrix, but not opportunistic fungi, e.g.: Candida species, can resist the effects of the active oxygen radicals released during the respiratory burst (Schaffner A et al., 1986).
• “Shape shifters”. Ability to grow in different tissue forms facilitates tissue invasion. e.g.: Histoplasma is dimorphic and the yeast forms multiply within host macrophages.
• Thermotolerance. Pathogenic fungi can grow at 37oC.

Using these stratagems, fungi are able to withstand host defenses. Fungi are ubiquitous in nature so that human and animal exposure is common but disease is uncommon and linked to: (a) host factors, outlined above, and (b) in the case of primary respiratory pathogens the inhaled dose of infectious propagules.

Example. The demonstration of fungi in blood drawn from an intravenous catheter may correspond to colonization of the catheter, to transient fungemia (i.e., dissemination of fungi through the bloodstream), or to a true bloodstream infection. The physician must decide which fits the clinical status of the patient based on physical examination, laboratory tests, and imaging studies. The decision to treat is not trivial, because systemic fungal infections require the aggressive use of drugs, some with considerable toxicity.

Are fungal diseases communicable? Most mycotic agents are soil saprobes and mycoses are generally not communicable from person-to-person. Some exceptions are dermatophytes: Tinea pedis exposure in gym locker rooms, scalp ringworm in young school children; oropharyngeal or vulvovaginal candidiasis and, probably, colonization with Pneumocystis jirovecii.
 

Mycotic disease outbreaks
Outbreaks occur when the environment containing primary respiratory pathogens is disturbed. These fungi have a particular, characteristic ecologic niche in nature. In this environment, the normally saprobic fungi proliferate and develop, providing a source of fungal elements and/or conidia, to which humans and animals, the incidental hosts, can become exposed. Illustrations are:

• Coccidioidomycosis among more than 300 participants of the World Championships of Model Airplanes, held each year in Lost Hills, Kern County, Calif., a highly endemic area.
  (Centers for Disease Control and Prevention, CDC, 2001).
• Histoplasmosis. At least 36 persons attending a Lung Association event in November 2007 at the Iowa Governor’s mansion contracted histoplasmosis. Bat droppings were found in the mansion's attic, in at least one other room, and in air filters. Bird and bat nesting sites and their guano are a known source of Histoplasma conidia.
• Blastomycosis. Six cases of blastomycosis occurred among 2 groups of 4th-6th grade students visiting a beaver pond at an environmental camp at Eagle River, Wisconsin in 1984. Cultures of soil from the beaver lodge and rotten wood near the beaver dam yielded Blastomyces dermatitidis (Klein et al., 1986).
• In recent years outbreaks of mycoses have accompanied the use of contaminated injectable medicines, insufficiently antiseptic contact lens cleaning and storage solutions.

The physician must be able to elicit a complete history from the patient including occupation, avocation, and travel history. This information is frequently required to raise or confirm a differential diagnosis.
 

Types of specimens received for direct examination and culture are similar to those submitted for bacteriology.
 

More Information

Specimen Processing
Chances to recover fungi are increased, with bacterial growth minimized, when clinical specimens reach the laboratory within 2 h of collection. This guide applies especially to urine specimens, which also may be stored at ≤ 24 h at 4 degrees C. Exceptions are that hair, nails, and skin scrapings may be stored up to 72 h at room temperature before culture, and they may be shipped by mail. CSF specimens are stored at 30oC and never refrigerated because CSF is a good culture medium and fungi will continue to replicate at room temp or 30oC.

Microscopic detection is facilitated when wet mounts of certain specimens are digested with 10% KOH ± Calcofluor: skin scrapings, hair, nails, corneal scrapings, and wound exudates. Visibility of fungal elements is increased when wet mounts are combined with fluorescent brightener, Calcofluor. Sputum specimens may require digestion with Mucolyse® (dithiothreitol in phosphate buffer) to reduce viscosity. Tissue obtained by biopsy or surgery is prepared for culture by grinding or homogenization if Histoplasma is suspected, but for other fungi and Mucorales species, mincing and/or finely slicing are preferred so as to not disrupt hyphal elements.

Histopathology
Formalin fixed, paraffin embedded (FFPE) tissues are treated with special fungal stains, or subject to fluorescent antibodies, to reveal fungal elements. The H&E stain does not always tint the organism, but it will stain inflammatory cells. The Gomori methenamine silver (GMS) stain is used to reveal fungi which stain black against a green background (figure 8). A combined H&E-GMS stain variation will demonstrate both fungi and the inflammatory response. See here.

Figure 9 shows asci (Cysts) of Pneumocystis jirovecii in lung tissue, stained with methenamine silver and hematoxylin and eosin (H&E). The walls of the cysts are stained black.

Other special fungal stains for FFPE tissue sections

• Periodic Acid Schiff is a good general stain for fungi, and can be counter-stained with hematoxylin to demonstrate tissue reactions.
• Mucicarmine will stain the capsule of Cryptococcus species.
• Fontana Masson stains fungal cell wall melanin.
• Fluorescent antibodies may be used for microscopy on fixed tissue sections but commercial reagents are scarce. One good example is the Merifluor® Pneumocystis kit (Meridian Bioscience, Inc.)
• FISH. Fluorescent in situ hybridization has found an application in smears of blood cultures. Fluorescent labeled peptide-nucleic acid probes are hybridized to fungal RNA. The AdvanDX® Peptide Nucleic Acid in Situ Hybridization Yeast Traffic Light system is available for microscopic detection of Candida species directly from blood culture bottles.

Culture
Definitive diagnosis requires culture and identification. Emmons modification of Sabouraud dextrose agar (SDA-Emmons) is extensively used for primary isolation of pathogenic fungi but Mucorales, black molds, dermatophytes, and yeast have higher percentage recoveries when plated on inhibitory mold agar (Scognamiglio et al., 2010). Cycloheximide may be added to suppress saprobic fungi but then a medium without cycloheximide should be paired. Chloramphenicol is often included to inhibit bacterial growth. If Histoplasma or Blastomyces are suspected an enriched medium such as brain-heart-infusion agar is indicated, bearing in mind that media supplemented with cycloheximide will inhibit the yeast form of these dimorphic fungi. There are many other formulations of mycologic media.

Sources of such formulations can be found here. This site is originated by Prof. Lynne Sigler of the University of Alberta Microfungus Collection and Herbarium, Canada. The Difco™ & BBL™ Manual of Microbiological Culture Media” (2nd Ed.) can be viewed and downloaded from the bd.com website.

Cultures of primary systemic dimorphic fungi, (e.g.: Blastomyces, Histoplasma) are identified in the laboratory by (a) slide culture and/or tease mount methods revealing characteristic microscopic morphology and (b) importantly by DNA probe tests of their mold forms grown at 25-30 degrees C (AccuProbe, Hologic Inc., San Diego, CA), (c) Recently MALDI-TOF (see below) has become an important, rapid, and reliable tool for identification.

Fungi grow best at 30 degrees C but if that temp is not available 25 degrees C will suffice. It is unnecessary to incubate an additional culture at 37 o C unless there is reasonable suspicion of a thermally dimorphic fungus. Cultures are not considered negative for growth until after 4 weeks’ incubation. Once a pure culture is obtained identification is made by observing colony and microscopic morphology for molds. Rapid tests for yeast identification are summarized in Reiss et al., 2012 section 2.3.7.2.
 

Identifications based on DNA sequence and proteomics analysis
The field of medical mycology reliant on classical morphologic diagnostic methods is now fully engaged in automated sample processing and walk-away instrumental analysis leading to a diagnosis from either isolated colonies or, identification directly from patient blood samples or from positive blood cultures. Such methods are expensive, but often do not require highly trained technologists, and shorten the time to a result to hours, or even less time.

MALDI-TOF MS
Matrix Assisted Laser Desorption/Ionization-Time-of-Flight-Mass Spectrometry. With minimal sample preparation from small isolated colonies MALDI-TOF MS produces a spectrum of protein fragments of known molecular mass. The resulting peptide mass fingerprint leads to faster, even same day, clinical decisions. When an incubated sample becomes a visible colony (~ 105 cfu) it can be subject to laser analysis, taking less than 10 min. An organism ID can then be made with little or no reduction in reliability. An end-to-end system including a database is the BioTyper® (Bruker Daltonics).

Commercial Multiplex PCR Assays for rapid detection of fungal infections
MicroSeq® Rapid Microbial Identification System (Life Technologies Inc.) combines reagents, instrumentation and a database of curated D2 18S rDNA sequences for upwards of 800 yeasts and molds. Alternative databases exist e.g.: MycoBank at the International Mycological Association, and GenBank at the National Center for Biotechnology Information (NCBI).

Luminex® xMAP Fungal Assay. This is a PCR micro-bead probe fluid array (Luminex Molecular Diagnostics) that detects Candida species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, C. guilliermondii, and C. krusei) or a mold 11-plex panel. To understand the method please view the MAGpix video.

FilmArray® (BioFire Diagnostics, A bioMérieux Company) is an FDA approved multiplex PCR with integrated sample preparation and automated analysis that detects 5 Candida species in positive blood cultures: Candida albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis. Sample from a positive blood culture is injected into a sample pouch and then the process then is automated and, including sample preparation, takes approximately 2 min to a result. Software calls and reports either a positive or negative for each microbe in the array blood panel. Detection is via a fluorescent dye binding to double stranded DNA. Microbes are identified depending on which well in the film array is positive. An illustrative video explains the method.

T2 Candida (FDA approved) (T2 Biosystems) Magnetic resonance technology is used to detect PCR amplicons of Candida DNA isolated from whole blood. Cells from 5 different Candida species in whole blood can be detected without the need to incubate blood samples. The T2Dx® instrument is automated: extracting DNA from whole blood followed by PCR amplification of rDNA. Detection is via nanoparticles coated with DNA probes complementary to different Candida species. A signal is generated and detected by T2 magnetic resonance. Nanoparticles with superparamagnetic properties increase magnetic resonance signals. These particles are coated with Candida species-specific probes. NMR spectrometers are available as benchtop models. The assay identifies Candida albicans and/or C. tropicalis, C. parapsilosis, and C. glabrata and/or C. krusei. The test does not distinguish between C. albicans and C. tropicalis. The test does not distinguish between C. glabrata and C. krusei. Details of the method may be viewed in the submission to the U.S. FDA.

IRIDICA assay (IBIS/Abbot Molecular) (not available in the USA). This multiplex PCR method directly detects microbial DNA in blood. It is capable to detect C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. This system is not available in the U.S.A. The PCR target is large subunit rDNA for ~200 fungal species.
Automated sample processing extracts DNA from 5 mL of whole blood. Hundreds of diverse microbes are identified from the species-specific genetic signatures in the PCR fragments using electrospray ionization mass spectrometry (PCR/ESI-MS). A computer parses and reports detection of 673 species of bacteria and Candida on the basis of multi-locus amplicon base composition signatures. The method can identify microbes from uncultured blood in less than 8 h.


Blood cultures
Detection of fungemia is an essential part of laboratory identification. A positive blood culture requires an immediate report to the physician of a presumptive fungal infection, with specific identification to follow. Two general types of blood culture methods are in use: manual and automated. Manual methods consist of broth, broth with an agar layer, or a solid agar medium. Examples of manual methods are (1) A biphasic broth/agar combination, the Septi-Chek® paddle device, using slide agars immersed in broth (BD Diagnostic Systems, Sparks, MD). (2) The Isolator® solid medium, system (Alere N.A., Waltham MA) is a single tube test combining lysis and centrifugation: lysis of RBC and WBC releases phagocytosed yeast forms of dimorphic fungi, then centrifugation through a dense fluorocarbon cushion produces a sediment which is planted to agar medium.

Automated continuously monitored blood culture systems. There are choices of such systems which have become the standard method in large medical centers. They are based either on the emission of CO2 during respiration of fungi or measurement of head gases above the surface of the broth blood culture bottle.

Blood culture bottles in the BacT/ALERT® (BioMérieux Corp.) system change color when the pH changes with increasing CO2 concentrations. The BD Bactec™ FX blood culture (BC) system has dye in the sensor at the bottom of the vial which reacts with CO2 released by microbial growth. This modulates light absorbed by a fluorescent material in the sensor. A photo detector then measures fluorescence and turns “on” indicating growth in the bottle. VersaTREK™ is another automated system that measures head gas changes in the blood culture bottles when there is growth.

Serology

Antibody tests
Serology may be helpful when applied to a specific fungal disease. The efficacy of serology varies with different mycoses. The serologic tests will be discussed under each mycosis. The most common serologic tests for fungi are based on double immunodiffusion, complement fixation and enzyme immunoassays (EIA). Double immunodiffusion and complement fixation usually detect IgG antibodies. Some EIA tests detect both IgG and IgM antibodies.

Antigen Detection
Several antigen detection methods have received good confirmation in clinical practice and are generally available in the U.S.A.

• Cryptococcal antigen test in CSF, plasma, or serum (CrAG® LFA, IMMY Corp, Norman, OK). Uses a lateral flow “dipstick” test and is rapid, sensitive and, with sample dilutions, can indicate a titer.
• Galactomannan antigenemia is detected in invasive aspergillosis (Platelia™ Aspergillus EIA, Bio-Rad.com). See here.
•  Histoplasma polysaccharide antigen test (HPA) is a good indicator of active invasive or disseminated histoplasmosis. MiraVista Diagnostics, Indianapolis IN
• Screening serum test for fungi in general: (1→3)-ß-D-glucan detection (Fungitell® Assay, Associates of Cape Cod Inc.) Exceptions are the Fungitell assay does not detect this polysaccharide in fungi in the genus Cryptococcus, or in the order Mucorales.
  
  Skin testing (dermal hypersensitivity)
  This was popular as a diagnostic tool, but is now discouraged because the skin test may interfere with serologic studies, causing false positive results. It may still be used in clinical immunology to evaluate the patient's immune status and, where reagents are available and approved for use, as a population exposure index in epidemiologic studies.
   

MOLECULAR STRUCTURE
Amphotericin B 
Ketoconazole 
Griseofulvin  
5-fluorocytosine 

MOLECULAR STRUCTURE

Ergosterol

Caspofungin

This introduction to antifungal agents describes the class of the agent, its mode of action, and a summary of its action spectrum. Further information is included in discussion of the individual mycoses. Structure diagrams for antifungal agents can be found here.  

Although one of the first anti-infective agents (oral iodides) was an anti-mycotic first used in 1903, the development of antifungal agents lagged the development of antibiotics against bacteria. Discovery of compounds with selective toxicity for the invading fungus avoiding serious adverse effects to the host has proved difficult because mammals and fungi are both eukaryotic, with similar cellular organelles and biochemical pathways. For example, an important drug target is membrane sterols, ergosterol in the fungal cell membrane and cholesterol in the mammalian cell membrane. Binding of drug to the ergosterol pathway carries risk for concomitant damage to host membranes. Fortunately, the armamentarium now has more choices and less toxicity.

Polyene antimycotic agents
The natural products, nystatin and amphotericin B (AmB), were co-discovered in 1953 from a Streptomyces nodosus soil isolate from Venezuela. Structure analysis revealed AmB has 7 conjugated double bonds linked to an amino sugar, mycosamine. It is “amphipathic” with a lipophilic face and a hydrophilic face, the latter containing 6 hydroxyl groups. The 2-faced structure is important in its mode of action. AmB is potent and fungicidal but is light labile and water-insoluble. Squibb laboratories in 1958 devised a way to obtain a suspension with sodium deoxycholate solution. The lyophilized product added to a glucose solution forms a micellar suspension that can be infused into patients. Nystatin is too toxic for IV use and is reserved for mucosal and skin applications. AmB has acute effects after IV administration: thrombophlebitis, fever, chills, nausea. Because of these and dose-limiting nephrotoxicity, in the 1980s AmB was incorporated into liposomes consisting of dimyristoyl phosphatidylcholine and dimyristoyl glycerol in a lipid-drug wt ratio of 12:1 to form liposomal AmB. This formulation has reduced nephrotoxicity and fewer infusion-related reactions. Other polyenes were discovered but aside from these two only pimaricin (natamycin) is in clinical use.

Mode of action. AmB binds to ergosterol in the fungal membrane producing ion channels through which cell contents leak resulting in cell death. A secondary mode of action occurs from auto-oxidation of AmB inducing oxidative stress that may contribute to rapid fungicidal activity.
Action spectrum. The major applications for AmB and liposomal AmB are invasive candidiasis, cryptococcosis, mucormycosis, and treatment for mycoses caused by dimorphic endemic fungal pathogens.

Azole antifungal agents
These are chemically synthesized based on the imidazole structure, moving later to triazoles with broader action spectra. Clotrimazole and miconazole were introduced in the late 1960s. Adverse effects and unpredictable pharmacokinetics limit clotrimazole to topical treatment. Miconazole is a useful topical agent but toxicity limits its parenteral use. Ketoconazole is an imidazole which, for almost a decade, was the only available oral agent for systemic mycoses. Shortcomings were inter-patient variation, poor CNS penetration, fungistatic nature, with adverse effects including drug-induced hepatitis, inhibition of testosterone and cortisol. Moreover, it was associated with poor response rates and recurrences of major mycoses. The era of triazole antifungal agents began with fluconazole in 1990. It is water soluble, can be delivered IV and, after oral administration, absorption is almost complete. It enters the CSF with concentrations nearing 80% of serum levels. Fluconazole came into wide use for mucosal and invasive yeast infection. The need for a broader spectrum azole was met in 1992 with the introduction of itraconazole, active against dimorphic endemic fungi and Aspergillus. The debut of extended spectrum triazoles followed, voriconazole in 2002 (based on the structure of fluconazole) and posaconazole in 2006 (similar in structure to itraconazole).

Mode of action. Azoles inhibit ergosterol biosynthesis at the C-14 demethylation stage. Through their azole ring, they form a complex with the heme iron of P-450 demethylase. Depletion of ergosterol results in cell membrane damage with collateral damage to membrane-bound enzymes active in nutrient transport, chitin synthesis, and growth. Azoles are fungistatic against yeast species, e.g.: Candida and Cryptococcus, but other triazoles appear fungicidal against Aspergillus spp.

Action spectrum. Fluconazole is active against most Candida and Cryptococcus spp., Histoplasma capsulatum, Coccidioides spp., and Paracoccidioides brasiliensis. It is in general use for maintenance therapy of AIDS-cryptococcosis. Itraconazole is used with good results against yeast and molds especially dermatophytes, Sporothrix schenckii, and for endemic dimorphic pathogens, against non-life threatening, non-meningeal forms of disease. Voriconazole is recommended for primary treatment of invasive aspergillosis; with AmB and liposomal AmB reserved for initial and salvage therapy where voriconazole cannot be used. Voriconazole is important in treating Fusarium mycosis, as well as AmB-resistant species: Aspergillus terreus and Pseudallescheria boydii. Posaconazole is active against opportunistic molds including the difficult to treat Mucorales and is approved for prophylaxis of invasive fungal infections in stem cell transplant recipients, and those with hematologic malignancy and prolonged neutropenia. Owing to the development of these triazoles, the treatment of invasive fungal infections is no longer limited by acute toxicity.

5-fluorocytosine (5-FC)
This is rarely used as monotherapy because of rapid development of secondary resistance. Combined with AmB it is useful in induction therapy for cryptococcal meningo-encephalitis.

Mode of action. Transported into fungal cells by a cytosine permease, host cytosine deaminase converts 5-FC into 5-fluorouracil (5-FU). After phosphorylation and incorporation into RNA, miscoding and disruption of protein synthesis ensues. Also, phosphorylated 5-FU is converted to its deoxynucleoside and blocks DNA synthesis by inhibiting thymidylate synthase.

Allylamines
Terbinafine is a lipophilic agent in the allylamine class of drugs.
Mode of action. Terbinafine inhibits an early step of ergosterol biosynthesis, the squalene epoxidase enzyme, resulting in squalene accumulation which increases membrane permeability and disruption of the fungal cell.

Action spectrum. Terbinafine concentrates in the skin and nails after oral administration and has a role in treating dermatophytosis including nail infections. Topical formulations are sold over-the-counter. Terbinafine combined with itraconazole or voriconazole may be synergistic in treating particularly resistant melanized molds, such as Lomentospora (was Scedosporium) prolificans. An explanation for this is that the two agents block ergosterol at different points in its biosynthesis.

Echinocandins
This class of natural products has a cyclic hexapeptide structure linked to an acyl lipid side chain. Anidulafungin discovered in 1974, is a semi-synthetic modification of a product from Aspergillus nidulans and was finally licensed in 2006. Caspofungin was approved in 2001 and micafungin in 2005. They are generally safe with few drug interactions. All three echinocandins were developed for daily IV infusion.

Mode of action. Echinocandins are noncompetitive inhibitors of the plasma membrane-bound β-(13)-D-glucan synthase enzyme complex, interfering with synthesis of fibrillar β-(13)-D-glucan, a cell wall component of Candida and of several other fungi. Without this glucan fungal cells become osmotically fragile. Mammalian cells lack this polysaccharide so that echinocandins have low host toxicity and reduced adverse effects.

Action spectrum. All three echinocandins are equally active as first line therapy against candidemia and invasive candidiasis; they are cidal for Candida spp. and fungistatic for molds. With respect to aspergillosis, caspofungin is recommended only for salvage therapy. Echinocandins lack activity against: Cryptococcus species, agents of the dimorphic endemic mycoses, and the difficult to treat molds: Fusarium, Scedosporium, and Mucorales.

Griseofulvin
A natural product of Penicllium griseofulvum, in 1952 it was recognized as the “curling factor” producing distorted fungal hyphae. Dermatophytes were very sensitive to griseofulvin but not yeast or bacteria. The key structural feature of this unusual molecule is the spirobenzofuranone moiety. Oral bioavailability is variable because it is poorly water-soluble.

Mode of Action. Griseofulvin inhibits mitosis strongly in fungal cells and weakly in mammalian cells by affecting mitotic spindle microtubule function. After oral administration, griseofulvin deposits in keratin precursor cells with greater affinity for diseased tissue. The drug binds to new keratin which becomes resistant to fungal invasion. Once the keratin-griseofulvin complex reaches the skin site, it binds to fungal microtubules (tubulin) altering fungal mitosis.

Action spectrum. Griseofulvin is a second line drug for the treatment of ringworm of the skin, hair, and nails caused by Trichophyton or Microsporum species. It is little used today because other drugs are more rapid acting with greater efficacy. Adverse effects of griseofulvin are usually mild including self-limited hepatotoxicity but very rare severe reactions are known: toxic epidermal necrolysis and related Stevens-Johnson syndrome, exacerbation of lupus.

Topical antifungal therapy
The preference for systemic or topical therapy of mucocutaneous and cutaneous mycoses depends on the immune status of the host and the type and extent of infection. Infections with dermatophytes, Candida spp. and Malassezia spp. yeasts can be treated topically with a variety of creams, lotions, ointments, powders, and spray forms. Terbinafine, ketoconazole, or miconazole in creams or sprays are used for tinea pedis, tinea cruris. Nystatin powder is used for intertriginous candidiasis and ketoconazole cream for seborrheic dermatitis associated with Malassezia spp. Oral clotrimazole troches, miconazole slow release tablets, or nystatin suspension or pastilles are used to treat oropharyngeal candidiasis. Vaginal candidiasis may be treated with creams or pessary form of clotrimazole, or nystatin. Refractory dermatophytosis such as tinea unguium and tinea capitis respond better to systemic therapy. Esophageal and vulvovaginal candidiasis respond well to systemic fluconazole therapy.
 

Literature Cited

 

 

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