Mycology Dermatophytes Please click audio icon to hear Carol’s narration Mycology Dermatophytes Hi I’m Carol Larson, your guide thru this tutorial about the dermatophyte molds. This lesson will be of value to you as you rotate thru the Mycology unit during 2nd semester. You will find an audio icon on each screen of this presentation. Please click on the icon to hear my narration for that specific screen. You may also follow along with the lecture handout entitled “Dermatophytes” that you can print out from this blackboard lesson. Division of Medical Technology Carol Larson MSEd, MT(ASCP)

Basic Characteristics Click icon for audio Basic Characteristics Medium growth rate (1-3 weeks) Identification Colony morphology Microscopic morphology Hyphae – hyaline & septate Macroconidia, Microconidia Physiological tests Clinical significance – Tinea (ringworm) The dermatophytes as a group have a medium growth rate of 1-3 weeks. Key characteristics used to identify this group of molds is to evaluate colony morphology, microscopic morphology, and physiological tests. Microscopically we look for the hyaline, septate hyphae, and the presence or absence of macroconidia and microconidia. The clinical significance of the dermatophytes is that they can cause Tinea. This is a Latin word for “worm” or “ringworm” and is an infection that involves the superficial areas of the body, including the hair, skin and nails. The Dermatophytes break down and utilize keratin found in these superficial areas as a source of nitrogen (but they are unable to penetrate the subcutaneous tissue so the disease is limited to just these areas.  

Clinical Significance Click icon for audio Clinical Significance Skin Tinea corporis Tinea pedis Tinea cruris Body sites that can be affected by the dermatophytes is limited to skin, hair and nails. When skin is infected, different areas of the body where infection occurs has a specific term. Tinea corporis is ringworm of the body and any of the common dermatophytes may be involved in this type of infection. You can see an example of this in the right-hand picture. Tinea pedis is ringworm of the feet, otherwise known as athlete’s foot, and Tinea cruris is ringworm of the groin area, also known as jock itch.

Clinical Significance Click icon for audio Clinical Significance Hair Tinea capitis Tinea barbae Ectothrix Endothrix When hair in affected, terms used include Tinea capitis, which is ringworm of the hair and scalp, and Tinea barbae known as ringworm of the beard. A couple of additional terms used that refer to the involvement of the hair shaft include: Ectothrix referring to conidia on the outside of the hair shaft. You can see an example of this on the lower-left. And Endothrix referring to conidia on the inside of the hair shaft as pictured on the lower-right.

Clinical Significance Click icon for audio Clinical Significance Nails Tinea unguium Nails can also be infected with dermatophytes and the term used for this is Tinea unguium, or ringworm of the nails.

Clinical Significance Click icon for audio Clinical Significance Dermatophyte Skin Hair Nails Microsporum X Trichophyton Epidermophyton The 3 main genus that we will focus on include Microsporum, Trichophyton, and Epidermophyton. From this table you can see which ones infect which body sites. An easy way to remember these is to remember that all three infect skin, Trichophyton having the prefix “tri” in its name infects all three body sites, Epidermophyton having the prefix “epiderm” focuses on skin and nails are an extension of skin. That leaves Microsporum who is the opposite of Epidermophyton and infects hair.

Epidemiology Anthropophilic Zoophilic Geophilic Man Animals Soil Click icon for audio Epidemiology Anthropophilic Man Zoophilic Animals Geophilic Soil Dermatophytes are found worldwide and hang out in several locations. Some species are found primarily just in man and are known as Anthropophilic. Other species are found primarily in animals such as cats and dogs and are referred to as Zoophilic. With these, man can easily become infected when handling animals. Last, some species are found primarily in soil and known as Geophilic.

What three body sites do the dermatophytes infect? Hair, skin and nails

What is the difference between endothrix and ectothrix? Endothrix means the mold has conidia inside the hair shaft, whereas Ectothrix means the conidia are only on the outside of the hair shaft.

What infection do the dermatophytes cause? Tinea (also referred to as “ringworm”). Another term that can be used is dermatophytosis.

Laboratory Diagnosis Specimen collection Direct examination Culture Click icon for audio Laboratory Diagnosis Specimen collection Direct examination Culture Identification Now lets look at how we are able to diagnose Tinea in the lab. We will focus on specimen collection, direct examination, culture and identification of the dermatophytes.

Specimen Collection Hair Skin Nails Transport in sterile petri dish Click icon for audio Laboratory Diagnosis Specimen Collection Hair Plucked, not cut, from edge of lesion Skin Wash, scrape from margin of lesion Nails Scrapings from nail bed or infected area Transport in sterile petri dish When hair, skin and nails are collected to be evaluated for fungal infections, there are very specific requirements in how to collect the specimen. When collecting hair, the hair should be plucked, not cut, and from the edge of the lesion. It is important to choose hairs that fluoresce under a Wood's lamp or, if there is no fluorescence, to choose broken or scaly ones. The hair is then place into a sterile petri dish and transported to the lab. Skin should first be washed well, and then scraped from the outer margin of the lesion onto folded black paper. This aids in seeing the scrapings. You can then place the skin scrapings into a sterile petri dish. Finger nails and toe nails must be scraped from the nail bed or from infected areas after the outer layers are discarded. The scrapings are then placed into a petri dish.

Direct Examination Examine hair for fluorescence Laboratory Diagnosis Click icon for audio Laboratory Diagnosis Direct Examination Examine hair for fluorescence Wood’s lamp Yellow green fluorescence = positive IF the primary care provider suspects that a patient’s hair is infected, he/she may utilize a wood’s lamp to check for fluorescence of the hair shafts. A positive test shows bright yellow-green fluorescence of the hair. There are a couple of Microsporum species (canis and audouinii) who are positive. A negative test would show no fluorescence. Microsporum gypseum and the Trichophyton species are negative.

Direct Examination Examine specimen for fungal elements Click icon for audio Laboratory Diagnosis Direct Examination Examine specimen for fungal elements 10% KOH preparation Calcofluor white stain When the specimen arrives in the lab, it is often beneficial to perform a direct examination of the clinical material. This can be done in a couple of ways: A 10% KOH preparation can be used to detect the presence of fungal elements in hair, skin and nails. The KOH dissolves the tissue and debris leaving just the fungal elements. Another procedure we can do is a Calcofluor white stain. This is a fluorescent stain that aids in the detection of fungal elements by taking up the fluorescent stain. The picture provided here shows a positive Calcofluor white stain showing arthroconidia and hyphae.  

Specimen processing Hair Nails Laboratory Diagnosis Click icon for audio Laboratory Diagnosis Specimen processing Hair Cut into short segments Nails Mince into small pieces Hair must be cut into short segments before setting up a culture. Also nails must be minced into small pieces before culturing. Skin scrapings are used as collected for setting up cultures.

Culture Media Select two media types Antibiotics Laboratory Diagnosis Click icon for audio Laboratory Diagnosis Culture Media Select two media types General purpose – Sabouraud’s agar Selective – Mycosel agar Antibiotics Gentamicin: inhibits normal bacterial flora Cycloheximide: inhibits saprophytic fungi Each specimen is divided between at least two types of culture media – a general purpose fungal media such as Sabouraud’s agar, and a selective media for fungal pathogens such as Mycosel agar. The use of antibiotics in the media such as gentamicin will inhibit the overgrowth of bacteria and incorporation of cycloheximide will prevent the overgrowth of the rapidly growing saprophytic fungi. Please review your Mycology Introduction Lecture notes for more information about other fungal culture media.

Culture Growth Requirements Click icon for audio Laboratory Diagnosis Culture Growth Requirements Place specimen pieces on culture media Can streak for isolation Incubate at 30°C in ambient (room) air Growth at 3 days to 3 weeks Examine plates frequently for 4 weeks The cultures are incubated at 30°C in ambient air. There is one exception and that is Trichophyton verrucosum that requires 35ºC. Visible growth can occur as soon as 3-4 days, with mature growth between 1-3 weeks but the cultures must be examined frequently for 4 weeks.

Identification Colony morphology Microscopic morphology Click icon for audio Laboratory Diagnosis Identification Colony morphology Microscopic morphology Scotch tape preparation Tease prep Slide culture Once growth has been detected on the culture plates, the colony morphology and microscopic morphology are evaluated. For the microscopic examination, the three standard procedures used in Mycology can be performed to begin the identification process of the mold. These are the Scotch tape preparation, the tease prep and the slide culture. The Scotch tape prep and the tease prep are a quick way to identify at least to genus level of the mold. The slide culture must be incubated for at least 7 days before examining the slide. This method enables the technologist to identify to the Genus level and possibly species level. Many clinical labs will just report to the Genus level for the dermatophytes.  

Identification Physiologic tests Laboratory Diagnosis Urea hydrolysis Click icon for audio Laboratory Diagnosis Identification Physiologic tests Urea hydrolysis Hair perforation Rice grain media Vitamin requirements Physiologic tests are sometimes used to help in the identification of the dermatophytes. This is a way to speciate the dermatophytes. Examples of these tests include Urea hydrolysis in which the mold is Incubated for 2-3 days on Christensen’s Urea Agar and look for a pink color. This test helps to differentiate between Trichophyton rubrum (negative) and Trichophyton mentagrophytes (positive). Another test to help differentiate between these two is the hair perforation test. Sterile uninfected hair is incubated at 25ºC with the mold for 10-14 days in a moist chamber. The hair is then observed microscopically for the presence or absence of conical perforations of the hair shaft. Trichophyton rubrum is negative and Trichophyton mentagrophytes is positive. Other tests not included in your objectives include growth on rice grain media and vitamin requirements. Please be aware that several other physiologic tests exist to aid in speciation of the dermatophytes.

How can hair, skin and nails be evaluated directly for fungal elements? Wood’s lamp fluorescence (hair only), 10% KOH preparation, and Calcofluor white fluorescent stain.

What are the incubation requirements when suspecting a dermatophyte infection? Fungal media is incubated at 30°C in ambient air for 4 weeks. There is one exception and that is Trichophyton verrucosum that requires 35ºC.

What primary procedures are performed to identify the dermatophytes? Colony morphology, microscopic morphology (Scotch tape prep, tease prep, or slide culture), and physiologic tests such as urea hydrolysis and hair perforation.

Etiologic Agents Microsporum species Epidermophyton species Click icon for audio Etiologic Agents Microsporum species Epidermophyton species Trichophyton species Dermatophyte Skin Hair Nails Microsporum X Trichophyton Epidermophyton Now we will next focus on some of the specific dermatophytes that cause Tinea. As I mentioned earlier, the three genus of interest are Microsporum, Epidermophyton, and Trichophyton species. They all cause ringworm, but have differences in the body sites they like to infect as this table reflects.

Microsporum canis Colony morphology: Click icon for audio Lets start with Microsporum canis. Its usually grows at about 1 week. Its colony morphology is usually membranous with a feathery periphery and its center is white to buff over an orange-yellow. The reverse side of the plate will show a lemon yellow or yellow-orange color.

Microsporum canis Microscopic morphology: Click icon for audio Microscopically, Microsporum canis has very characteristic macroconidia. They are thick-walled, spindle-shaped, multiseptate, rough-walled, and some have a curved tip. Microconidia are rarely seen. Microsporum canis can cause Tinea capitis and tinea corporis, often from coming in contact with dogs and cats. Infected Hair will fluoresce under a Wood’s Lamp.

Microsporum gypseum Colony morphology: Click icon for audio Microsporum gypseum colony will grow at 1 week. Its colony morphology is cinnamon-colored and powdery. Its reverse shows a light tan and can eventually take on a lavender color as seen here.

Microsporum gypseum Microscopic morphology: Click icon for audio Microsporum gypseum’s microscopic examination has thick-walled, rough, elliptical, and multiseptate macroconidia. When comparing to Microsporum canis, gypseum’s walls are a little thinner and the tips are more rounded. The microconidia are few or absent. Microsporum gypseum can cause Ectothrix Tinea capitis. Infected Hair will NOT fluoresce under a Wood’s Lamp.

Microsporum audouinii Click icon for audio Microsporum audouinii Colony morphology: Microsporum audouinii will grow at 2 weeks. Its colony morphology is a Downy texture with white to salmon-pink color. Its reverse is tan to salmon-pink.

Microsporum audouinii Click icon for audio Microsporum audouinii Microscopic morphology: For Microsporum audouinii the microscopic exam can be quite interesting. It has sterile hyphae with terminal chlamydospores, Favic chandeliers, and pectinate bodies. These pectinate bodies look like little combs. Macroconidia are rarely seen and appear bizarre-shaped. Microconidia are rarely seen or absent.   Microsporum audouinii can cause epidemic Tinea capitis in children . Infected hair will fluoresce under a Wood’s Lamp.

How can Microsporum species be differentiated from each other microscopically? Characteristic appearance of the macroconidia, and the general appearance of the hyphae (such as pectinate bodies). As a group, Microsporum have few to absent microconidia.

Epidermophyton floccosum Click icon for audio Epidermophyton floccosum Colony morphology: Now lets look at Epidermophyton floccosum. It grows at 1 week. The center of its colony tends to be folded and is khaki green in color with a periphery of yellow. On the reverse you see a yellowish brown color with observable folds.

Epidermophyton floccosum Click icon for audio Epidermophyton floccosum Microscopic morphology: Epidermophyton floccosum has macroconidia that are large, smooth-walled, and multiseptate. The can be borne singly or in clusters of two or three on the hyphae. Very important to remember is there are NO microconidia with Epidermophyton floccosum!   Epidermophyton floccosum can cause Tinea cruris and tinea unguium.

Trichophyton rubrum Colony morphology: Click icon for audio The last genus we will look at is Trichophyton. There are two species that are most often seen so we will focus on just these two. Lets first look at Trichophyton rubrum. It grows at 2 weeks. It colony morphology varies from white downy to pink granular and rugal folds are common. Its reverse is yellow when young; however, a wine red color commonly develops with age (thus its name rubrum).  

Trichophyton rubrum Microscopic morphology: Click icon for audio Trichophyton rubrum is very non-descript microscopically. Macroconidia are usually absent, but when present are smooth, thin-walled, and pencil-shaped. Microconidia are very numerous as you can see in these pictures and are usually teardrop in shape and most commonly borne along the sides of hyphae.   Trichophyton rubrum can cause Tinea corporis, tinea pedis, tinea cruris, and tinea capitis.

Trichophyton rubrum Physiological tests Urea: negative Click icon for audio Trichophyton rubrum Physiological tests Urea: negative Hair perforation: negative Trichophyton rubrum is differentiated from Trichophyton mentagrophytes by doing a urea hydrolysis and hair perforation test. Trichophyton rubrum is urea negative and hair perforation negative.

Trichophyton mentagrophytes Click icon for audio Trichophyton mentagrophytes Colony morphology: Trichophyton mentagrophytes grows at 7-10 days. Its colony morphology can vary greatly. It can have a texture of downy, granular, or velvet. The color is white and occasional has a light yellow periphery in younger cultures. Its reverse is buff to reddish brown. Downy Granular Velvet

Trichophyton mentagrophytes Click icon for audio Trichophyton mentagrophytes Microscopic morphology: Microscopically Trichophyton mentagrophytes is somewhat similar to T. rubrum and difficult to differentiate. Macroconidia can be numerous or rare depending upon strain, and are thin-walled, smooth, club-shaped, and multiseptate. Microconidia are plentiful, round, and most commonly borne in grapelike clusters or laterally along hyphae. Spiral hyphae are present in about 30% of isolates. Trichophyton mentagrophytes can cause Tinea corporis, tinea capitis, and tinea barbae.

Trichophyton mentagrophytes Click icon for audio Trichophyton mentagrophytes Physiologic tests: Urea: positive Hair perforation: positive Trichophyton mentagrophytes is differentiated from Trichophyton rubrum by doing a urea hydrolysis and hair perforation test. Trichophyton mentagrophytes is urea positive and hair perforation positive.

How can Trichophyton mentagrophytes be differentiated from Trichophyton rubrum? Urea hydrolysis and hair perforation tests. T. mentagrophytes is positive for both, and T. rubrum is negative for both.

How can Microsporum, Epidermophyton, and Trichophyton species be differentiated microscopically? Microsporum has numerous thick-walled macroconidia with RARE microconidia, Epidermophyton has numerous club-shaped macroconidia hanging out in groups of 2-3 with NO microconidia, and Trichophyton has thin-walled macroconidia and MANY microconidia.

In Summary … Causes Tinea (ringworm) Medium growth rate = 1-3 weeks Click icon for audio Dermatophytes In Summary … Causes Tinea (ringworm) Medium growth rate = 1-3 weeks Grows on Mycosel agar Identification Colony morphology, microscopic exam, and physiologic tests Etiologic agents Microsporum, Epidermophyton, Trichophyton species Now to quickly summarize what we have focused on in this lesson. The dermatophytes are a group of molds that cause the cutaneous infection known as Tinea or ringworm. They are able to infect only skin, hair and nails. When cultured they have a medium growth rate and will grow on Mycosel agar that contains antibiotics that inhibit bacteria and the saprophytic molds. To identify the dermatophytes, we evaluate their colony morphology, microscopic morphology, and physiologic tests such as urea and hair perforation. We learned how to identify specific Microsporum, Epidermophyton and Trichophyton species that we commonly see in the Mycology lab.

Who am I? Microsporum canis Potato Dextrose Agar Reverse LPCB Stain of Slide Culture Microsporum canis

Epidermophyton floccosum Who am I? Potato Dextrose Agar LPCB Stain of Slide Culture Epidermophyton floccosum

Trichophyton mentagrophytes Who am I? Hair Perforation Potato Dextrose Agar LPCB Stain of Slide Culture Trichophyton mentagrophytes

Who am I? Microsporum gypseum Potato Dextrose Agar Reverse LPCB Stain of Slide Culture Microsporum gypseum