Introduction to Medical Mycology

By Itabangi Herbert Learning objectives To understand the defining characteristics of fungi To understand the defining characteristics of the six phyla of fungi To understand the morphological classification of fungi To learn to differentiate between sexual and asexual reproduction To learn the diseases caused by different fungi To learn the laboratory methods employed to diagnose fungal infections. To learn some control methods of fungal diseases

Kingdom fungi (mycota)

Six subdivisions (phyla) known a. Myoxomycota b. Chrytridiomycota c. Oomycota d. Zygomycota e. Ascomycota f. Basidiomycota All medically important fungi belong to only three of the above subdivisions ie.Zygomycota, Basidiomycota, and Ascomycota Mycology is the study of fungi. The diseases caused are called mycoses. Mycoses are chronic infections because fungi grow slowly. Fungi are saprophytic or parasitic eukaryotic microorganisms, distinct from plants and animals. In other words- To define the exact limits of the fungal groups in a few words is virtually impossible, however generally speaking Fungi are eukaryotic, heterotrophic, unicellular to filamentous, rigid cell walled, spore bearing organisms that usually reproduce by both sexually and a sexual means. Further more they are insensitive to antibiotics;

Eukaryotes

- In sense that they contain membrane organelles and exhibit mitosis

Heterotrophs - In sense that they are achlorophyllus and therefore not autotrophic like plants and algae but rather they are heterotrophic absorptive organisms that are either saprophytes (living on dead organic matter) or parasitic (utilizing living tissue) Importance of fungi Fungi are important both negatively and negatively; Fungi are abundant in the human environment. They are common causes of damage to crops, foods stuffs, fabrics, building materials. etc They can be harmful not only to the material needs of the humans but also to individuals. Fungal diseases are primary infections or allergies, but they may also be caused by eating food stuffs contaminated by fungal toxins (mycotoxins) However Fungi do not rank with the protozoa, helminthes, viruses, or bacteria as causes of human suffering. They may be very common, but they seldom kill. Unlike cholera, malaria or yellow fever, they do not cause wide spread or dangerous epidemics. They can nevertheless be a cause of major distress or disability and affected individuals can have their livelihoods or their lives seriously threatened. In 1975 and 1976 the WHO (World Healthy Organization) acknowledged that mycoses are a serious medical and social problem through out the world, and attempts should be made to provide mycological information and the services to areas where they are a public health problem. The widest range of fungal infections and the most serious are to be found in tropical and developing countries. Structure of fungi Fungi occur in two basic forms or stages a) Unicellular or yeast form which is defined morphologically, as a single called fungi that reproduces by simple budding to form blastoconidia. Colonies on media are usually moist, pastae or mucoid with alcoholic smell. Yeast like fungi may be Basidiomycetes such as Cryptococcus neoformans or Ascomycetes (Endomycetes) such as Candida

albicans b) A filamentous or mould form which is a vegetative growth of filaments. Structures such as mushrooms consist simply of a number of filaments packed tightly together, and reproduction is by spores or conidia c) Dimorphic fungi Some fungi especially the pathogenic species may exhibit two forms of growth and are called dimorphic fungi .they can grow either as filamentous fungi or (moulds) or yeasts. Dimorphism is temperature dependent. At 37C the fungi is a yeast and at 25C its a mould. Moulds produce a great variety of conidia which are borne on specialized hyphae or conidiophores. Many moulds can be identified by the morphology of these spores and by their arrangement on the hyphae

Two basic types of reproduction are found in fungi. Sexual reproduction- This involves fusion of two nuclear that then generally undergo meiosis. This method involves plasmogamy (cytoplasmic fusion of two cells), Karyogamy (fusion of two nuclear), genetic recombination and meiosis and the resultant is sexual spore which can be zygospores, ascospore and basidiospores. A sexual reproduction- This is a mode of reproduction which arise following mitosis of a parent nucleus. Conidia arise either by budding of conidiogenous hyphae or by differentiation of preformed hyphae.

Fungal pathogenicity.
The ability of fungi to cause disease appears to be an accidental phenomenon with the exception of a few dermatophytes; pathogenicity among the fungi is not necessary for the mentainace or dissemination of the species. More than 50,000 spp of fungi have been described. They are classified into several major classes according to their cellular organization and the ways they reproduce.

The principal mycoses (fungal infections) affecting man can be distinguished by the sites of the body affected, each type has a different pattern of disease characteristics. The two major physiological barriers to fungal growth with in the human body are temperature and redox potential most fungi are mesophilic and can not grow at 37 C similarly most fungi are saprophytic and their enzymatic pathways functions more efficiently at the redox potential of non living substrates than at the relatively more reduced state of living metabolizing tissue. In addition, the body has a highly efficient set of cellular defences to combat fungal proliferation. Thus, the basic mechanism of fungal pathogenicity is its ability to adapt to the tissue environment and to with stand the lytic activity of the hosts cellular defences. In general, the development of human mycoses is related primary to the immunological status of the host and environmental exposure, rather than to the infecting organism. A small number of fungi have the ability to cause infections in normal/healthy humans by;

having a unique enzymatic capacity, exhibiting thermal dimorphism having an ability to block the cell-mediated immune defences of the host

There are then many opportunistic fungi which cause infections almost exclusively in debilitated patients whose normal defense mechanisms are impaired. The organisms involved are cosmopolitan fungi which have a very low inherent virulence. Currently, there has been a dramatic increase in fungal infections of this type, in particular Condidiasis, Cryptococcosis, Aspergillosis, and Zygomycosis. More recently described mycoses of this category include hyalohypomycosis phaeohyphomycosis. All together, some 200 human pathogens have been recognized from among an estimated 1.5 million spp of fungi.

Clinical groups for fungal infections

Skin mycology. Superficial mycoses Cutaneous mycoses Subcutaneous mycoses

Infections Disease mycology Dimorphic systemic mycoses Opportunistic systemic mycoses.

Basic morphological and clinical classification of medically important fungi Fungi

Moulds

Yeasts

Aseptate hyphae

septate hyphae

Basidiomycete Without pseudohyphae Systemic

with psenudo hyphae endomycetes Cutaneous

- Cryptococcus

neofornmans -Geotrichum -melassezia albicans -guiramondi -stellodea Subcutaneous -basidriobolus spp -conidiobolus Rhinosporidium seeberi Systemic Rhizopus spp Absidia spp Mucor spp superficial -microsporum spp -Trichophyton spp -Epidemophyton spp -Malassezia fur fur -piedraia hortae -Trichosporon beigelii Superficial Malassezia fur fur Subcutenous - Phialophora spp - Cladosporium spp -Madurella spp -Acremonium spp -Petriellidium boydil -Phialophora jeanselmi -leptos phaeria senegalensis. systemic -Histoplasmma spp -Blastomyces dermatitids -paracoccidiodes brasilensis monomorphic Dimorphic -melassezia candida -

Skin mycology
(a) Superficial /Cutaneous mycoses These affect the skin, hair, and nails. They are confined to the body surfaces and dont directly involve living tissues, i.e. they are keratinophilic. The important superficial mycoses include; i) Ring worm (dermatophytosis) These are caused by Microsporum spp, Trichophyton spp and Epidermophyton flocossum ii) iii) Pitryriasis versicolor (Tinea versicolor) caused by malassezia fur fur Piedra (Trichosporosis) caused by predraia hortae and Trichosporon beigelii

Ring worm fungi / Dermatophytosis / Tinea. Superficial and cutaneous fungi are often grouped together since they infect the same areas of the body, i.e. skin, hair and nails These mycoses are non invasive and basically a symptomatic, involving just the top keratincontaining layer of the skin or hair while cutaneous mycoses (dermatomycoses) affect the deeper epidermal layer, producing more tissue destruction and symptoms. Primary superficial/cutaneous mycoses are caused by candida spp for example C.albicans, C.stelloidea, C. guillarmondi, C.krusei etc or the dermatophytes for example M.canis,M.audouinii,M.gypseum,M.ferrugeuim,T.mentagrophytes,T.tonsurans,T. verruncosum,T. Violaceum, T.rubrum and Epidermophyton flocossum The term Tinea, or Ringworm, has been applied traditionally to the diseases elicited by dermatophytes. However it has been suggested recently that this term be replaced by the term dermatophytosis, followed by body area affected e.g. Tinea Capitis should be replaced by dermatophytosis of the scalp. If the skin is abraded, systemic pathogens, for example coccidiodes immitis, also may rarely produce cutaneous infections without involving the rest of the body. In addition, systemic

organisms may exhibit secondary cutaneous manifestations as part of the disseminated disease process. Secondary infections therefore must be carefully differentiated from primary ones, as the prognosis and treatment are quite different. The disease process in dermatophytosis is unique for two reasons: a) No living tissue is invaded the keratinized stratum corneum is simply colonized. However, the presence of the fungus and its metabolic products usually induces an allergic and inflammatory eczematous response in the host. The type and severity of the host response is often related to the species and strain of dermatophytes causing the infection. b) The dermatophytes are the only fungi that have evolved a dependency on human or animal infection for the survival and dissemination of their species. The Dermatophytes can be divided into 3 broad epidemiological groups.
GEOPHILIC

These dermatophytes normally inhabit the soil where they are believed to decompose keratinaceous debris. Some species may cause infections in animals and man following contact with soil (e.g. Microsporum gypsum). These dermatophytes are primarily parasitic on animals. Infections may be transmitted to man following contact with animal host (e.g. Microsporum canis)

ZOOPHILIC

ANTHROPOPHILIC

These dermatophytes are primarily parasitic on man and have only rarely been known to infect animals, presumably following contact with man (e.g. Trichophyton rubrum)

NOTE: Anthropophilic fungi such as Microsporum audouinii, Trichophyton rubrum, Trichophyton tonsurans and Trichophyton Violaceum are unable to colonize animals other than man. They are the only fungi that have developed a dependency on man for the maintenance and dissemination of their species. Malassesia furfur Malassezia furfur formally called Pityrosporum Orbicularis

Pathogenicity This organism causes Pitryriasis versicolor (tinea versicolor) an symptomatic skin infection characterized by scaly patches of different colors; reddish brown, brown, and white (dipigementation/hypopigementation) the lesions tend to fluorescence yellow orange under woods light. Note; M.furfur can be mould as yeast like but both forms may co-exits. Its therefore not truly dimorphic (b) Sub cutaneous These are called mycoses of implantation because they are acquired when the pathogen is inoculated through the skin by minor cuts or scratches or by thorns as in splinter wounds. The principal subcutaneous mycoses are: i) ii) iii) iv) v) vi) Chromomycosis caused by phialophora spp and cladosparium carrionii Mycetoma caused by several spp of fungi and actinomycetes Subcutaneous phycomycosis, caused by basidiobolus or conidiobolus spp Rhinosporidiosis, caused by Rhinosporidium seeberi Lobomycosis Subcutaneous zygomycosis

Subcutaneous mycoses cont These are chronic, localized infections of the skin and subcutaneous tissue following the traumatic implantation of the aetiologic agent. The causative fungi are all soil saprophytes of regional epidemiology whose ability to adapt to the tissue environment and elicit disease is extremely variable. Chromoblastomycosis / Chromomycosis. This is a chronic warty mycosis of the skin and subcutaneous tissues caused by phialophora spp e.g. P. pedrosoi, P. dermatitidis. The lesions are usually on the lower leg but other parts of the body can also be affected. The term mossy foot is sometimes used when the foot is affected.

Mycetoma. A chronic granulomatous disease of the subcutaneous and deep tissues. Progressive destruction of tissue leading to loss of function of affected site. The foot is commonly affected (madura foot) but other parts of the body can also be involved including the hands, head, thigh and walls of the chest. Mycetoma is caused by both fungi and bacterial actinomycetes. When caused by fungi the swelling is called Eumycetoma and by actinomycetes its called actinomycetoma. Note: Its very important to know the origin of the Mycetoma whether fungal or bacterial simply because actinomyetic infections respond to treatment with antibiotics and sulphonamides while fungal infections are resistant to ant antimicrobials

Infectious Disease mycology.

(c)Dimorphic systemic mycoses The systemic mycoses are also referred has deep mycoses. They are acquired by inhalation and may spread from the lung and involve any part of the body, Wide spread infections can be fetal. Skin lesions are often present. These are fungal infections of the body caused by dimorphic fungal pathogens which can overcome the physiological and cellular defences of the normal human host by changing their morphological form. They are geographically restricted and the primary site of infection is usually pulmonary, following the inhalation of conidia. Systemic mycoses include. Histoplasmsis, coccidioidomycosis, Blastomycosis, paracoccidioidmycosis etc. Histoplasmosis Caused by Histoplasma capsulatum and histoplasma duboisii. Transmission; By inhalation of spores from soil contaminated with the excreta from bats, chickens or other birds Distribution; Worldwide distribution especially USA.

It may be a symptomatic, acute, or chronic. The acute pulmonary form produces symptoms similar to pneumonia whereas the chronic form resembles pulmonary tuberculosis. Generalized H. capsulatum infections are rare, occurring mostly in infants and patients with depressed immunity. H. duboisii is often referred to as large-cell Histoplasmosis became the intracellular yeast forms are three to four times larger than H. capsulatum. Infection with H. duboisii may take the form of a localized skin nodule which may ulcerate, bone abscess, or a generalized infection. The lungs are only occasionally infected. Blastomycosis B. dematitidis causes Blastomycosis. Although formerly thought to occur only in North America, in recent years blastomycosis has been recognized in tropical and subtropical African countries. Following inhalation of the spores, B. dermatitis is can cause chronic lung disease resembling pulmonary tuberculosis. Occasionally, an infection spreads to the central nervous system (CNS), urogenital system, bone, subcutaneous tissue, and the skin. Cutaneous infections can lead to ulceration or warty granuloma. Paracoccidioidmycosis P. brasilensis causes paracoccidioidmycosis, a systemic mycosis that is found Brazil also other parts of S. America and Central America. This infects the mucous membranes of the mouth and the nose where it causes painful ulcerative granulomas with destruction of nasal and month tissue. The lymph glands of the neck may also become involved and the infection can then spread by way of the lymphatic system to the hugs, spleen, and other organs of the body.

(d)Opportunistic systemic mycoses These are caused by opportunistic pathogens, harmless in its normal habitat but can become pathogenic in a host that is debilitated, traumatized, under treatment with broad spectrum antibiotics or whose immune system is suppressed by drugs or

immune disorders.
Opportunistic fugal infections of the body occur almost exclusively in debilitated patients whose normal defense mechanisms are impaired. The organisms involved are cosmopolitan fungi, which have a very low inherent virulence. The increased incidence of these infections and the diversity of fungi causing them has paralleled the emergence of AIDS and the use of antibiotics, cytotoxins, immunosuppressives, steroids as other macro disruptive procedures that result in lowered resistance of the host. Principal opportunistic mycoses include Candidiasis Cryptococcosis Aspergillosis Mucormycosis (Zygomycosis)

Mycoses in the neutropenic patients. In these patients virtually any fungus that can grow at 37C and gain access to the blood stream may cause disseminated infections. These infections pose enormous problems in terms of initial clinical recognition, diagnosis and laboratory identification of the causative agent. The prevalence of serious fungal infections in these patients is constantly increasing especially candidiasis. Mycoses in AIDS patients The emergence of AIDS has been responsible fore a dramatic increase in human infections caused by the yeast-like fungi, candida and Cryptococcus. Mucosal candidiasis occurs in almost all AIDS patients with C.albicans accounting for greater than 85% of the infections Cryptococcal infection in patients with AIDS is considered incurable, and life long therapy is necessary to suppress the infection. Almost all infections are caused by C. neoformans var. neoformans. Infact AIDS is the classic setting for this fungus which has the ability to inhibit phagocytosis and survive the non specific inflammatory immune defects of the host Candidiasis (moniliasis)

Clinical manifestation of candidiasis may be acute, subacute or a chronic to episodic. Involvement may be localized to the mouth, throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, or the gastro intestinal tract (GIT) or become systemic as in septicemia, endocarditis and meningitis. In healthy individuals, candida infections are usually due to impaired epithelial barrier function and occur in all age groups, but are mostly in the newborn and the elderly. They usually remain superficial and respond to treatment readily. Systemic candidiasis is usually seen in patients with cell mediates immune deficiency, and those receiving aggressive cancer, immunosuppression, or transplantation therapy Several species of candida may be aetiogical agents, most commonly, candida albicans and rarely C. tropicalis, C. Krusei, C. parapsilosis, C. guillermondi C. Kefyr, and C. glabrata. All are ubiquitous and occur naturally on humans. Cryptococcosis This is a Chronic, sub a cute to a cute pulmonary, systemic or meningitic disease, initiated by eh inhalation of basidiospores and or desiccated yeast cells of C. neoformans primary pulmonary infections have no diagnostic symptoms and are usually sub clinical. On dissemination, the fungus usually shows a predilection for the central nervous system, however skin, bones and other visceral organs may also be come involved. Although C. neoformans is regarded as the principal pathogenic species, Cryptococcus albidus and C. laurentii have been implicated in human infection. C. neoformans is an encapsulated basidiomycetes yeast-like fungus which has been divided into two varieties, C. neoformans var. neoformans and C. neoformans var. gatti. C. neofomans var. neoformans (A and D serotypes) have a worldwide distribution is now the most significant world wide opportunistic pathogen in humans especially AIDS patients. Natural sources of Cryptococcus; these include dung of caged avian birds, pigeon excreta, rotting vegetables, fruits, wood and diary products.

Nutritional adaptations Fungi usually grow better in an environment with a PH of 5.0,which is too acidic for growth of most bacteria. Most moulds are aerobic, so they grow on surfaces rather than throughout the substrate. Most fungi are more resistant to osmotic pressure than bacteria and are therefore able to grow in high sugar or salt concentrations. Fungi can grow on substances with low moisture content ,too low to support growth of bacteria. Fungi are capable of metabolizing complex carbohydrates e.g. leginin, and can grow in unlikely substrates e.g. painted walls, shoe leather, discarded news papers.

Laboratory Diagnosis of Fungal Infections

The successful diagnosis and treatment of fungal infections is very much dependent on a team work approach which inevitably involves communication between a large number of individuals ranging from infections disease physicians (IDPs), Microbiologist, Oncologists, Histopathologists and various scientific and technical Laboratory staff. Clinical maternals Skin and nails scrapings Hair follicles Urine Sputum Bronchial washings Cerebrospinal fluid (CSF) like in Cryptococcus meningitic cases Pleural fluid

Blood Tissue biopsies from various visceral organs and indwelling catheter tips Pus etc.

Laboratory Specimen Processing In general, direct microscopy and culture should be performed on all specimens received by the Laboratory Microscopy provides vital information, often an immediate presumptive diagnosis is possible, which is of particular importance in the immunosuppressed patient. Microscopy usually consists of either (a) wet mounts in 10% KOH with Parker ink, or India ink, (b) smears for Gram, Giemsa and PAS staining, and (c ) histopathology of tissue sections using H&E, Gomori methenamine silver (GMS) and PAS. Note: candida and Aspergillus may be missed in H&E stained sections, therefore GMS stained sections are essential for tissue pathology. Routinely, cultures should be maintained for one month. Cultures should be examined regularly, fungal growths identified and significant isolates reported as soon as possible. Clinicians also need to be aware of the following laboratory requirements in order to generate an appropriate specimen for a diagnosis. 1. A relevant clinical history. This information is vital to the laboratory; (a) it ehnhances processing of the specimen which in some cases may increase the likely isolation of some fungi, especially zygomycetes; (b) it aids in the determination of significance of fungal isolates, especially those from no-sterile sites such as sputum, bronchial washings and skin; and (c ) it is the only way of alerting laboratories that they may be dealing with a potential pathogen. The safety of laboratory staff will depend on this information as cultures of dimorphic fungi like Histoplasma capsulatum represent a severe biohazard and must be handled with extreme caution in an appropriate pathogen handling cabinet. 2. An adequate amount of suitable clinical material. Unfortunately many specimens submitted are either of an inadequate amount or are not appropriate to make a definite diagnosis. The laboratory needs enough specimen to perform both microscopy and culture. Too often specimens are either placed totally in formalin, thus allowing no culture or alternatively they are not sent to histopathology. 3. At present, serological tests, with the possible exception of antigen detection for

Cryptococcus neoformans, are of limited value and must be supported by clinical, cultural and other evidence. 4. With the exception of histopathology, routine laboratory turn around time for the isolation and identification of fungi and sensitivity testing may take several weeks. Even for candida albicans, this may take up to 5 days, 8 days for other yeasts and common moulds like Aspergillus, and often 2 to 4 weeks for other filamentous moulds. Although histopathology offers the most rapid confirmatory diagnosis it is not always possible to obtain suitable biopsy material or to make a specific identification of the fungus. Basically, the clinician will need to make a clinical diagnosis and commence antifungal treatment well in advance of any laboratory confirmation. Control of fungal infections Tretment of infected individuals Proper disposal of avain dung Public health education Avoid sharing clothes with infected individuals Personal hygiene

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