Mycology Practical Questions and Answers by sammyc2007

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									Mycology Questions 2007: Feedback 1) Dark hyphae are one reason for a fungal isolate having a dark appearance macroscopically (e.g with the phaeohyphomycetes), but sometimes hyalohyphomycetes have a dark thallus even though their hyphae is colourless. Explain. A key feature in the early differentiation and identification of fungi is  determining which ones have pigmented hyphae. The fungi that have pigmented  hyphae usually have a dark colony, but not always. On the other hand, plenty of  fungi have dark colonies, especially dark green even though their hyphae are  hyaline and colourless. This is because pigmentation of conidia also contributes  to the colour of the colony.  Bear in mind that conidia pigmentation is of virtually no value in the  identification of fungi.  2) Explain how dermatophyte test medium is useful in detecting and identifying dermatophytes. How does the medium work? Are all fungi that grow on the medium and produce a red colour definitely dermatophytes? DTM can be used in a number of ways in the detection and identification of dermatophytes, and some of these are addressed in other questions and answers within this set of questions. I won't cover all the ways in which it can be used in my answer, but give you an example of a way in which it can really be useful. Suppose you isolate a fungus from a specimen in which you might reasonably expect to find a dermatophyte. If on microscopic examination of the culture, sufficient conidia are present to produce an identification, there is no need to then set up a DTM. But what if the microscopy is unrewarding? In that case, why not set up a DTM at the same time as you set up the other media that you hope will induce conidiation. Again, if these other media are able to induce sufficient conidiation to produce a microscopic identification, then the DTM (which should be positive by then if the isolate is a dermatophyte) will be helpful but still not really necessary. But what if the initial second batch of media still do not produce sufficient conidia to give an identification. At this point, if the DTM was negative, then it would be reasonable to conclude that the isolate was probably not a dermatophyte and cut your losses, but if the DTM was positive, it would be worthwhile selecting some different media to try and induce conidiation.

To be useful, DTM must be selective and differential. Most students mentioned that DTM contains antibacterial and antifungal (saprophytes) agents, but not many mentioned the actual differential agent. Phytone is a relatively complex polypeptide that most organisms are unable to digest. Since dermatophytes have enzymes able to break down keratin, then these same enzymes presumably also allow the dermatophytes to break down the phytone polypeptide. The metabolic endproducts of polypeptide metabolism are usually alkaline, and in this case produce a red colour with the pH indicator. Although it is relatively uncommon it is possible for DTM to produce false positives. This may be because a particular non dermatophyte fungal isolate is resistant to cyclohexamide or is able to slowly overcome the inhibition, and is also able to hydrolyse phytone, or, it may be because you have a fungus that can overcome the inhibitory effect of the cyclohexamide and a small amount of bacterial growth that might provide the pH change required to produce a red colour. In this context it is possible for a dermatophyte to give a false negative if bacterial contamination of the slope is able to undo the pH change produced by the dermatophyte. Don't overdo this issue about bacterial contaminants producing false results. You need to have a pretty unusual set of circumstances coinciding before it will happen. First you must have bacterial contamination which is unlikely from a subculture from a primary selective fungal plate and needs the bacteria to be resistant to whatever antibacterial agent in the primary medium and the DTM, and then you need the bacterial growth to sufficiently change the pH to overide the change induced by bacterial growth. So, a red DTM is not definitely a dermatophyte. BUT SO WHAT. The result is not used in isolation, but in combination with other results such as microscopy of the isolate which would "suss" out any false positives. DTM is widely accepted as a sufficiently sensitive and specific medium for initial differentiation of dermatophytes from non dermatophyte fungi. There has recently been some debate regarding the performance of the original DTM medium, with concern about lack of specificity. A number of studies have shown that a number of non dermatophyte fungi can overcome the concentration of cyclohexamide and not only grow, but also produce a red colour change similar to that produced by dermatophytes. Salkin et al (1997) have produced a new medium, dermatophyte identification medium (DIM) that is reported as having superior specificity to DTM without any loss of sensitivity. DIM has a slightly different substrate, uses bromcresol

purple as a pH indicator and has a higher concentration of cyclohexamide. The exact formulation was not published presumably because of the intention to patent the medium and sell it commercially. The published results from Salkin et al are impressive. Of 223 dermatophytes tested, only one did not produce a visible alkaline shift with the bromcresol purple. The sensitivity was greater than 99%. However, 15 of 73 non dermatophytes that were able to grow on the DIM were able to also cause an alkaline pH shift. This initially gives a specificity of only around 95%, but since all 15 problematic non dermatophytes were clearly dematiacious, they are easily recognised as non dermatophytes on the basis of colonial morphology. When the reaction on DIM is considered in conjunction with colonial morphology, then the specificity approaches 100%. In both the media (DTM and DIM), I have only vaguely referred to the substrate. In DTM, the substrate is phytone, and in DIM it is neopeptone). Both these substrates are peptones; that is they are a heterogeneous mixture of different size polypetides formed from the enzymic hydrolysis of proteins. Phytone comes from soy protein, whereas neopeptone comes from animal protein. So is there any reason why the different media use different peptone sources? I don't know, but I suspect the change was more to do with making DIM sufficiently different from DTM to make it possible to patent the new medium. The same might be true of the change from phenol red to bromcresol purple. Maybe the only real functional difference between the media is the concentration of the cyclohexamide? 3) In some laboratories, the Sabs (or Sabs chlor) and the Mycobiotic are used routinely on all specimens, but on specimens with a positive direct microscopy, the DTM medium is included in the primary media - what is the rationale behind this approach? So you have grown a fungus from a skin scraping! Is it a dermatophyte? The fastest way to find out is to examine the microscopic morphology, and with a bit of luck it will be typical for a particular dermatophyte, and not only will you be able to identify it as "a dermatophyte", you will also be able to give it a specific name. But what if the isolate is not being cooperative and is not producing sufficient conidia on which to make an identification? Quite often, all you get on initial examination of a fresh isolate is "sterile hyphae". Subculturing onto other media

to stimulate conidiation is an obvious next step, although it may take 2 to 3 weeks to have sufficient growth for microscopic examination. One way of dealing with this is to subculture the organism onto a DTM slope and look for a colour change. The only problem being that this will take a week or so to go positive. If you want to have a faster answer, why not include a DTM slope with the primary media inoculated initially? This would give you an answer at the same time as you saw growth on the mycobiotic agar. The problem with this is that you probably only grow dermatophytes from about 5% of the specimens that you culture. This means that for every 5 DTM slopes that are useful, 95 are wasted. In a cost conscious environment, this may not be acceptable. There would be less waste if it were possible to identify (prior to culture) those samples that were more or less likely to contain a dermatophyte. This is achieved by doing direct microscopy, and on those that are positive (and therefore very likely to have a dermatophyte) you include a DTM, and on those that are negative (less likely to have a dermatophyte) you don't. As you know, a negative direct microscopy does not mean that you will not grow a dermatophyte; but using this strategy of media use, you will need to subculture a possible dermatophyte isolate onto DTM to find out. I wonder (well actually I don't wonder, but I'm wondering on your behalf) if the specificity and sensitivity of DTM (or DIM) would be different when used directly on clinical material as opposed to being used on subcultures from primary plates? I think they would both be worse, and it is up to you to think about why. 4) Would these organisms (Scopulariopsis and Fusarium)) be recovered if nail specimens were only cultured on media containing cyclohexamide. No. Scopulariopsis brevicaulis and Acremonium spp. are in fact hyaline hyphomycetes, and classified taxonomically in the same broad group of fungi as Penicillium spp. and Aspergillus spp. These are the kinds of "common contaminant" fungi for which cyclohexamide is included as an inhibitory agent to make culture media selective for dermatophytes. So, if you get "dermatophyte tunnel vision" and only culture specimens on media containing cyclohexamide, then there is a risk that some non dermatophyte pathogens may be missed.

Dermatophyte "tunnel vision" is how I refer to the common misconception that all "tinea" like lesions are caused by dermatophytes. They are not, and this applies especially to onychomycosis where non dermatophytes are well recognized.

5) Are there any clinical features that might suggest either Scopulariopsis or Acremonium as the causative agents of a particular case of onychomycosis? Nails infected with Scopulariopsis brevicaulis tend to develop a brown colouration and crumbly texture, whereas those infected with Acremonium spp. tend to be whitish. Like all such clinical findings, they are useful hints, but not diagnostic. Would you be able to differentiate (sometimes, but not always) a nail infected with Acremonium spp from a nail infected with Scopulariopsis brevicaulis or a nail infected with a dermatophyte? If your answer is NO, then you should and revise the online tutorial. 6) Similarity to which other very common hyaline fungus (which is a common contaminant of laboratory cultures) often leads to cultures of Scopulariopsis being prematurely discarded as not significant? What are the major distinguishing features? The conidiophore of Scopulariopsis is sometimes referred to as a "penicillus" which means "brush like". Isn't it a pity that the science (?art) of mycology seems obsessed with seemingly obtuse terminology ! What is wrong with simply saying "brush like"? Needless to say then, that there is some similarity between Scopulariopsis spp. and Penicillium spp. The most obvious microscopic difference being that the conidiophores of Scopulariopsis are shorter, the conidia larger and usually spiny or rough. 7) What are the individual purposes of the chloramphenicol and cyclohexamide? Chloramphenicol is a broad spectrum antibacterial agent, and cyclohexamide is a broad spectrum antimycotic agent. They can be used to suppress the growth of some bacterial/mycotic agents that may otherwise outgrow, obscure and therefore prevent the detection in culture of certain fungi from some clinical specimens.

There is no choice of antifungal (anti saprophytic fungi) agent, as only cyclohexamide inhibits the majority of saprophytes while NOT inhibiting the dermatophytes, but there is a choice of antibacterial agent. Some laboratories, or some culture media recipes actually use a combination of gentamicin and tetracycline. It doesn't really matter as long as the effect is broad spectrum bacterial inhibition. 8) Which medium would be most suitable for culturing clinical specimens for dermatophytes? Dermatophytes are commonly grown from specimens that are likely to contain numerous other organisms: both of a bacterial and fungal nature. They are also relatively slow growing, sometimes taking up to 4 weeks to become visible on first isolation, so it is necessary to use selective media. Since dermatophytes are known to be resistant to cyclohexamide, a culture medium containing both an antibacterial agent (such as chloramphenicol or gentamicin) and cyclohexamide would be most suitable. Even though the mycobiotic selective agar is considered the best for culturing dermatophytes, you need to remember that in many cases fungi other than dermatophytes (including fungi that are sensitive to cyclohexamide) may be involved. Classic examples are Candida albicans from either skin or nails, or Scopulariopsis brevicaulis from nails. This issue is further explored in question 14.

9) Are any of the class subclinical carriers of dermatophytes and which dermatophytes might you expect in this situation? In 2000, 2 isolates of T. mentagrophytes and 1 isolate of T. rubrum were isolated from students who were apparently asymptomatic. In 2001, one student was clearly symptomatic, and a dermatophyte was isolated, and one other student that gave a positive culture was asymptomatic. In 2002, one student grew a dermatophyte and he was actually symptomatic. It turns out that he actually had two different dermatophytes which in my experience is quite unusual.

This year (2006), no students were carriers, but one student was infected with E. flocossum. With asymptomatic dermatophyte infection/carrier states in humans, you would most definitely expect an anthropophilic organism. The organism must be well adapted to the host so that only a very weak host immune response is elicited. In this context it is important to stress the fact that the symptoms associated with dermatophytosis are usually due to the host response and not to any destructive enzymes or toxins that the fungus produces, nor to any invasion of living tissue since the dermatophytes only infect the outer dermis. Trichophyton rubrum is known to be capable of causing an asymptomatic infection/carrier state on the foot (in particular in the toe webbing) and this has been linked to the evolutionary adaptation of this previously tropical fungus to colder climates.

T.mentagrophytes has more than one variety: T. mentagrophytes var mentagrophytes is zoophilic and tends to have a more typical microscopic morphology, including spherical microconidia in dense clusters. This variety tends to be involved in tinea corporis. T. mentagrophytes var interdigitale is anthropophilic and is more often involved in tinea pedis and onychomycosis. The microconidia produced by this variety tend to be less spherical and more pyriform in shape and therefore look more like typical microconidia from a T. rubrum. If these strains produce spiral hyphae, then identification is easy, but if they don't, then further tests may be required to differentiate them from T. rubrum. It seems likely that the isolates of T. mentagrophytes from students in the year 2000 were var interdigitale.There was also another student in 2000 from which both E. floccosum and T. rubrum was isolated but this person was symptomatic. In 2001, the isolates from both students (symptomatic and asymptomatic were not fully identified, but on initial microscopy tended to resemble T. rubrum.

10) What is the purpose of KOH in direct fungal microscopy? How do you determine how long to digest for?

Direct microscopy is always important because it provides immediate information about the presence or absence of fungi in the specimen, and in some situations it might also provide some clues as to the identity of the organism. One of the problems with direct microscopy involving tissues infected with fungi is the relative sparsity of fungal elements. The relative thickness of skin scrapings and hair and nail clippings also makes it difficulty to see the relatively inconspicuous fungal elements. Potassium hydroxide (usually a 10 to 15% solution) can be used to digest or clear the specimen. The KOH effectively breaks down the keratinous tissue whilst not significantly affecting the chitinous fungal cell wall. After sufficient digestion, the sample can be squashed to make a thinner preparation for microscopy. The time required for sufficient digestion depends on the nature and thickness of the specimen. Fine pieces of skin might be sufficiently cleared after 5 to 10 minutes, whereas thick pieces of skin may require a couple of hours, and thick nail clipping may even require overnight digestion. Heating the KOH specimen mixture can speed up the digestion process as can the addition of some dimethyl sulphoxide (DMSO) which helps the KOH penetrate the specimen. One important precaution with digestion is not to over do it because although the fungal elements are relatively inert to the digestion process they will ultimately be destroyed which is why you cant use a digest for a permanent slide. To help visualise the fungal elements after digestion they can be stained with some blue or black ink. Lactophenol cotton blue tends to be decolourised by the alkaline conditions and should not be used. The ink can be added after digestion, or it can be added to the KOH to be used as a dual digestion/stain reagent. Note that all these preparations (stained or unstained) are wet preparations, in other words, you have the specimen and fluid (KOH etc) on a slide with a coverslip. 11) How reliable is the direct microscopic examination of skin and nail specimens in the diagnosis of dermatophytosis - which do you think is better, the PPV or NPV? Direct examination of skin and nail specimens for the presence of "fungal elements" is an established laboratory approach for the rapid diagnosis of

dermatophytosis. In general, this approach is quite specific in that there are not many situations in which fungal elements seen on direct microscopy are not due to the presence of a dermatophyte. Similarly, it is unlikely that non fungal structures (ie artefacts) are misidentified as fungal elements. So a positive test (detection of fungal elements) is usually associated with disease: ie good PPV. On the other hand, due to sparsity of fungal growth in some lesions/specimens and the relative frequency that small and indistinct fungal elements are missed, direct microscopic examination is relatively insensitive. So a negative test (fungal elements not detected on direct microscopy) does not exclude dermatophytosis (or any other fungal infection): ie poor NPV. In general, direct microscopy on skin specimens is probably both more sensitive and specific than similar tests on nail specimens. This is because the quality of specimen is usually better with skin scrapings than nail clippings, and the specimen is easier to clear with KOH, so that it is easier to see the fungal elements and there is less "rubbish" that may be mistaken for fungal elements. It should be noted that although dermatophytes are the most common cause of fungal skin and nail infections, there are other causes. Most notably, Malassezia furfur and Candida albicans for skin, and Candida albicans, Scopulariopsis brevicaulis, Acremonium, Aspergillus and Fusarium for nails. Although it may sometimes be possible to differentiate some of these causes on the basis of the direct microscopy, it is frequently impossible. In the absence of any other evidence, considering "fungal elements" to most probably be dermatophytes until proven otherwise seems a reasonable inference. 12) What are the advantages and disadvantages compared to culture? The advantages of direct microscopy are that a positive result is available in a couple of hours as opposed to 2-3 weeks. This means that appropriate therapy can be initiated sooner. This is particularly important in some cases because one of the best drugs for treating dermatophytosis (Terbanifine) is available (at normal cost as opposed to extra cost) only if there is supporting evidence of infection from the laboratory. When you think about this regulatory (and obviously cost saving) approach by the government, it must mean that the extra cost of pathology tests (to establish the diagnosis) is less than the cost of the extra drug that may otherwise be used on "non dermatophyte" and possibly "non fungal"skin conditions. The disadvantage of direct microscopy is that it is much less sensitive than culture, and even though direct microscopy is acceptably specific (ie few false

positives), it is still less specific than culture. In addition, a positive direct microscopy simply tells you that an infection is caused by a dermatophyte (most probably); it does not tell you which dermatophyte. Now, even though the specific identity of the dermatophyte has no direct bearing on the drug prescribed for treatment, the identity will have an impact on how the patient is managed. For example, do the family pets need to looked at or has the infection been acquired in some other way. It is worth looking at the special case of onychomycosis with respect to the sensitivity of direct microscopy compared to culture. In some studies, up to half of the infected nail clippings failed to grow on culture. In other words, direct microscopy of nail specimens might be about equally sensitive as culture. It is actually better to say "equally insensitive" as this reinforces the fact that especially with nail specimens, it is important to do both direct microscopy and culture.

13) What possible explanations are there for a positive direct microscopy and a negative culture? When answering this question, there is an assumption that the specimen is being analysed within the context of "skin, nail or hair sample being examined for the presence of a dermatophyte". In this context, if you have a positive direct microscopy and a negative culture there are 2 broad explanations. Firstly, it may be a false positive microscopy and a true negative culture; ie the patient does not have a dermatophyte infection. Alternatively, the direct microscopy may be truly positive and the negative culture a false negative; ie the patient does have a dermatophyte infection. Consider the possibilities for false positive microscopy. The fungal elements observed may have been artefact, or alternatively the fungal elements may have been non dermatophyte fungi that were present either as contaminants or colonisers, or indeed in some cases as pathogens albeit "non dermatophytes". There are more possibilities for a false negative culture. The patient may have been receiving some form of treatment that may hinder the ability to culture the organism although the fungal elements in the tissue would remain visible. It is also possible that the distribution of fungal elements is not consistent through the specimen and it is not unusual for "the best piece" to be used for

microscopy and then the remainder for culture. It is important to stress that if you only receive a small amount of specimen, it is probably best to use the majority (or even all) for culture since this is the most sensitive and specific test. It is also possible that despite the use of selective media, the presence of the dermatophyte in the culture is obscured by the overgrowth of bacterial and fungal contaminants. It is also possible (unfortunately) although hopefully not probable, that a dermatophyte is grown on culture but discarded because the individual handling the analysis fails to recognise the fungus as a dermatophyte. There are also a couple of other scenarios that do not fit neatly into the two broad categories just discussed. There are occasions when there may be positive direct microscopy but a "negative culture" that isn't really negative. For example some fungal skin/nail infections can be caused by "non dermatophyte" fungi, especially Candida albicans, which can cause both skin rashes and onychomycosis. In this context, if you only look for dermatophytes then there will be occasions when other fungi that may be pathogenic will be overlooked simply because they are not dermatophytes.

14) In what type of fungal infection of the skin is direct microscopy always more sensitive than culture? In tinea versicolor (caused by Malassezia furfur) the only reliable means of laboratory diagnosis is through direct microscopy. This is because the organism is very difficult to grow and certainly would not grow on the types of culture systems used for routine culture of skin scrapings. Since the culture will always be falsely negative (ie negative in the presence of infection), then direct microscopy must be more sensitive. This is also another example of the problem discussed in Q13. 15) It is good practise to always use a selective and non selective medium when culturing lesions thought to be caused by dermatophytes. Why ? If all dermatophytes are resistant to cyclohexamide then why not use just selective media (containing cyclohexamide) when culturing specimens from lesions suspected to be caused by dermatophytes? Well, this concept was alluded to in the last part of the answer to Q8. Because dermatophytes are the most common isolates from cutaneous lesions, it is hard not to develop "tunnel vision", or should that be "dermatophyte vision". Remember that other fungi, most of which are sensitive to cyclohexamide, can occasionally cause infections that resemble dermatophytosis. If we only used

selective media, then these fungi would not be detected, but having said that, it is no use growing these "other fungi", if the person is examining the cultures with "dermatophyte vision" and is not aware of which other fungi may be significant in certain situations.

16) All dermatophytes are resistant to cyclohexamide, so if a specimen gives fungal growth on the plain Sabs (or Sabs chlor) but not on the Mycobiotic, does this mean that the isolate is not a dermatophyte? Again, this question is about the possibility (as opposed to probability) that a dermatophyte may be unevenly distributed within a specimen. If you only grow a particular isolate on the Sabourauds agar (and there is no growth on the mycobiotic agar), then it could be that the organism was unevenly distributed. This is even more likely if you only have a very small amount of specimen and you have inoculated the "best" or "biggest bit" onto the Sabourauds, and only a "small" or "scummy" bit on the mycobiotic. This inevitably leads to the following question.If you only have a very small amount of specimen, say, only enough to culture on one type of medium, "do you culture only on plain Sabourauds (or Sabourauds with chloramphenicol but either way, a non fungally selective plate) so that you retain the chance of growing the full range of potential fungal pathogens, most likely a dermatophyte, but possibly something else, and in doing so, accept the risk that a dermatophyte (if present) might be overgrown by a fungal contaminant? Or, do you culture only on a mycobiotic, and maximise your chance of growing the dermatophyte (if present) because there is lower risk of overgrowth with fungal contaminants, and accept that if the pathogen is a "non dermatophyte" you have no chance of growing it? I don't have an answer (only an opinion), think about it yourself. 17) Both sporotrichosis and chromoblastosis are caused by dematiaceous fungi and in a broad sense are phaeohyphomycoses; the diseases are given distinct names not only because they are characteristic clinical entities but also because of the unique appearance of the fungal elements in the tissues. What are the distinctive fungal elements? With sporotrichosis, it is worthwhile remembering that the organism is dimorphic, and that a yeast phase is present in vivo. In this sense it would not fit the definition of a phaeohyphomycosis since there are no fungal hyphael

elements in the tissue. It is not unusual to see no fungal elements on direct microscopy, or sometimes you see only sparse round to elongated yeast cells. Classically (but rarely) you see the "asteroid" body, which is an individual yeast cell surrounded by eosinophilic staining host tissue. Again, with chromoblastmycosis, although it is not a true dimorphic fungus, there is an absence of hyphael forms in vivo. In the previous example of sporotrichosis, this tendency to form yeast cells in vivo was truly an organism specific phenomena, but this is not the case with chromoblastomycosis. Firstly, chromoblastomycosis can be caused by a number of different dematiacious fungi, and secondly, depending on the site and nature of infection, and the immunocompetence of the host, these same fungi can cause subcutaneous infection that is clinically distinct from chromoblastomycosis and may be associated with the presence of hyphael forms in the tissue. However, in true chromoblastomycosis, the characteristic tissue forms are "sclerotic bodies", which are brown, non budding, thick walled "yeast" cells. 18) Which other fungi are involved in chromoblastomycosis? Which other fungi are involved in eumycetoma? All fungi that cause chromoblastomycosis are dematiaceous. The most common are Cladosporium, Fonsecea and Phialophora. The fungi that cause eumycetoma can be either dematiaceous or hyaline. Most common examples are Madurella, Pseudoallescheria, Fusarium and Exophiala 19) Are all the agents of eumycetoma dematiaceous? Some cases of eumycetoma are actually hyalohyphomycoses - what does this mean? Eumycetoma is a fungal disease characterised by the clinical presentation and the presence of "granules in vivo", it is not classified on the basis of being caused by only one certain "type" or "group" of fungi in the way that the clinically miscellaneous mycoses, the "phaeohyphomycoses" and "hyalohyphomycoses" are classified simply on the basis that they are caused by dematiaceous and hyaline fungi respectively. If a fungal infection caused by a dematiaceous or hyaline fungus presents with the presence of in vivo granules, sinus formation and subcutaneous swelling, then it is a case of "eumycetoma".

20) What body sites are most commonly affected by these "traumatic implanation"mycoses, and can you suggest why they are more common in males than females. Usually hands and feet, as these body sites are more open to the types of traumatic events that might involve implantation of fungally contaminated material. The high male to female ratio of cases is a result of increased exposure in males rather than increased susceptibility to infection. 21) Because Candida albicans is the most commonly isolated pathogenic yeast, the germ tube is usually the first test performed on any yeast isolated. If the organism is germ tube positive, then it is reported as Candida albicans, but if it is germ tube negative the next step is not so clear. In some cases, you might feel comfortable with simply reporting Candida spp., but in other situations you might feel that it is more appropriate to use other tests to fully identify the isolate. Comment on these options. In answering this question, it is worthwhile outlining the commonly available  systems / tests available for the identification of Candida and other yeasts. The  germ tube is a simple test, and 95% or more of isolates of C. albicans are positive.  C. dubliniensis is also positive, but is an uncommon isolate and unlikely to be  encountered in a genital specimen. For germ tube negative isolates, morphology  of the yeast on cornmeal tween agar (Dalmau) plate can be helpful. The problem  with this is that it is a relatively old fashioned test, relies on the user having some  interpretive microcopy skills, is slow and will not differentiate C. albicans from  C. dubliniensis.  The new chromogenic plates are good at identifying C. albicans and the other  Candida spp which might be encountered in similar specimens.   Finally, there are other commercial “API like” kits that use a number of tests to  generate a profile number that can be compared with a database to give an  identification.  So for identification of yeasts, I think the following protocol is reasonable.  Any yeast that comes from a sterile site should be fully identified with a  commercial multi test kit. 

For yeasts that are grown from other sites (mouth, genital, skin, nail or other  mucous membranes) can be treated differently. A germ tube is a useful first test  because this test will rapidly identify Candida albicans which will be by far the  most common isolate. For germ tube negative yeasts, a chromogenic plate or a  commercial test kit should be used.  Alternatively, it may be more efficient to simply do a chromogenic test on all  yeast isolates (not sterile sites). This will be cost effective if you use the plate like  a DNAse plate and multi inoculate. The good thing about going straight to a  chromogenic plate is that it will reliably identify C. albicans and the other  common yeasts that are not albicans; in other words almost all the yeasts  encountered in routine settings.  Some laboratories have taken the approach that any yeast (with the correct  colonial and microscopic morphology) grown from a vaginal swab is C. albicans,  or alternatively, some laboratories do not bother to identify vaginal yeasts  beyond simply reporting them as Candida spp. The problem with these  approaches now is that non albicans species of yeasts are becoming increasingly  isolated from genital specimens, and the treatment can be different since some of  these other Candida species are more likely to be resistant to some of the  antifungal drugs.   

22) Briefly explain the principle behind Chromagar CANDIDA (CMA). In what ways is it superior to older more conventional methods of yeast identification? This medium incorporates a range of chromogenic substrates specific to certain  yeasts such that the most common yeasts (C. albicans, C. krusei, C. tropicalis and  C. parapsilosis) produce colonies of different colours. It is superior to the germ  tube in that it not only identifies C. albicans but also some of the other yeasts that  would otherwise simply be characterized as germ tube negative. It is superior to  the cornmeal tween agar morphology test as it is much easier to interpret,  although some laboratories find that the performance (specificity) of the CMA is  enhanced if it is combined with a cornmeal agar morphology test. It is superior to  the API multi test systems in that it is faster and easier to interpret, but is unable  to identify the enormous range of yeasts that those tests do. 

23) In body sites where Candida albicans is normal flora, what information can be used to establish - or form an opinion - on whether a particular isolate is involved as a pathogen or is simply a spectator? When considering ways to help determine the significance of Candida (or anything else for that matter) in such circumstances, it is worth remembering that it is seldom our responsibility to make the final decision. If you demonstrate the presence of a potential pathogen, report the fact and let the clinician make the final decision. We can, however, provide additional information that may assist the clinician. In the direct microscopy, the "amount" of organism present can be important. Did you see only occasional yeasts, or did you see large numbers? Possibly more important (this is debatable) is the nature of the fungal forms seen. There is an argument that suggests that the presence of yeasts and long hyphael forms correlates with invasiveness and pathogenicity. This doesn't mean that if you see only "yeasts" that the isolate is not causing disease, but it may mean that if you see hyphael forms, the Candida is more likely to be involved in a disease process. In culture, again, the amount of organism present is important. If you have only very light growth, then "innocent bystander" may be more likely, whereas if you have very heavy growth, then perhaps "presence as a pathogen" is more likely. In this context, it is important to consider that not only the amount of organism at the site of investigation, but the quality of the specimen collected, and the way it has been handled in the laboratory can also affect the amount of growth None of these considerations are hard and fast "rules", but in combination can be helpful in determining the significance of a given isolate.

24) Why is indian ink examination considered an essential component of CSF analysis in immunocompromised patients? Cryptococcal meningitis is not a common disease, so always performing a test to detect this organism may not represent the best use of resources. However, in sub-populations where the disease is more common, it is essential. Without an indian ink stain, the yeasts may be mistaken for host cells, particularly red cells since they are so refractile. In addition, in immunocompromised hosts, it is not uncommon for there to be no increase in

white cells in the CSF, or more commonly an increase in lymphocytes. Such findings may not alert the scientist to the presence of an infectious process, and the chance of an early diagnosis is missed.

25) In demonstrating phenol oxidase activity, why might caffeic acid be a more reliable substrate than "bird seed" and is it possible to use it in a more efficient way than incorporation into the medium? The problem with birdseed agar is that various batches of the special bird seen (common name "niger seed", real name Guizotia abyssinica) may contain different amounts of the substrate. In addition the extraction of the substrate (boiling and crushing the seed) before making the medium may vary in efficiency. This means that different batches of bird seed agar probably vary considerably in the content of the substrate, ultimately meaning that there may be great variability in the appearance of the organism on the media. Caffeic acid can also be used as a substrate, and because it is a chemically defined compound, the same amount can be used in the media and this will cut down on the variability of colonial pigmentation. It is possible to incorporate the caffeic acid into discs, and if these are placed on the plate (in the same way as optochin discs for example), then only the cryptococcal colonies around the disc are pigmented. This is a great method because it gets around the need to have birdseed agar plates in stock (they last for about 2 weeks) when you may only require the plates once a year. It would be very time consuming and cumbersome to have to make the plates from scratch when you needed them. With caffeic acid discs (which have a long shelf life), you can use standard media (sabs or BHI) and simply add the disc.

26) Can you suggest a possible reason if a seasonal fluctuation in cases of cryptococcosis occurred in Australia, but similar fluctuation was not apparent in other countries? Please be aware of the changes in the nomenclature of C. neoformans. C. neoformans var gattii has now been renamed as C. bacillisporus, and that of the two serotypes (A and D) of C. neoformans var neoformans, the A serotype has been renamed var grubii.

Firstly, the question assumes you realise that C. neoformans var neoformans has a worldwide distribution, but var gattii is really only found in certain parts of Australia and other limited areas around the world. So if the question is talking about a seasonal variation in disease incidence in Australia (as opposed to worlwide) then it is reasonable to assume that the seasonal change in incidence might be due to an Australian factor, which might be different seasonal exposure to C. neoformans var gattii. It is important to realise (and stress in your answer) that C. neoformans has a teleomorph (a sexual state), so that in the asexual state it is a yeast, but in the sexual state (a Basidiomycete called Filobasidiella) it is actually a different fungus from a morphological perspective, and reproduces by producing basidiopsores. It is postulated that the sexual state lives in a close relationship with certain types of gum trees, and that dispersal of the basidiospores occurs during the periods in which the tree is flowering. These basidiospores are possibly infectious for both man and other animal hosts, and presumably the basidiospore undergoes some type of transformation into the asexual yeast form once in the host. One student did pose an interesting scenario for possible increased risk of infection with var neoformans in summer. With this variety, the known types of exposure that are known to pose a risk of infection include exposure to the organism in dried pigeon (and other bird) droppings. In summer you would expect old pigeon droppings to be drier, and therefore more easily dispersed and inhaled. An interesting idea, although I am unsure of any studies or data that support the fact that infection with var neoformans has a seasonal bias. There has been a lot of work done recently on the association with various strains of Cryptococcus neoformans and particular environmental niches, and it is probably a much more complicated scenario than we would like. 27) The direct microscopic examination of clinical material for fungal elements is always an important aspect of laboratory investigation. It can be particularly important in zygomycosis in terms improving the outcome for the patient. Why? In many cases, fungal infections tend to be chronic or only slowly progressive in their clinical course. This is somewhat fortuitous since most diagnostic approaches (especially those that involve culture) tend to be rather slow.

A notable exception to this idea of slow progression, is rhinocerebral mucormycosis (a specific type of zygomycosis) which can be devastatingly destructive (or fatal) in a matter of days. Obviously a rapid diagnostic strategy is important in this situation. The very broad non septate hyphael elements are sufficiently typical, that seeing them in certain specimens is diagnostic of the involvement of a zygomycete. 28) In aspergillosis direct microscopy (ie microscopy on the specimen) can be important in determining the clinical significance of a given isolate. Explain. Aspergillus spores are essentially ubiquitous and are amongst the most common of fungal contaminants of culture plates. In addition, the organism is capable of colonising (ie growing but without overt host injury) in some body sites. Given these considerations, when you recover an Aspergillus in culture from a clinical specimen, what does it mean? Is it a laboratory or specimen contaminant, is it present as a coloniser, or is it a significant isolate associated with disease at the site the specimen was collected from? If you can demonstrate the presence of fungal elements consistent with Aspergillus in the direct clinical microscopy, then you can probably conclude that the isolate is "real", that is, it came from the patient and was not a contaminant. This evidence will probably not resolve the issue as to whether the isolate is a pathogen or a coloniser, but that's another story. 29) KOH preparations are standard for mycological examination of clinical material - what is calcafluor white and how is it an improvement on KOH? If you make a preparation of a specimen using a combination of KOH (for digestion) and calcafluor white and examine using a UV microscope, the sensitivity of detecting fungal elements is greatly increased compared to using KOH alone and phase contrast microscopy. It is generally easier to see a bright apple green fungal element against a dark background, than it is to see a slightly darker gray fungal element against other shades of gray background as you would see on plain phase contrast. The most important point to make here is that calcafluor white IS NOT an immunofluorescent stain. The binding of the stain to fungal cell wall is relatively specific, but it is not immunologically based.

30) If you were microscopically examining a biopsy specimen and saw a) broad segments of non septate hyphae, or alternatively, b) septate hyphae with dichotomous branching, what would these findings suggest? a) A zygomycete, and b) A hyaline hyphomycete (usually Aspergillus or less often a Fusarium). 31) When biopsy material is received for mycological analysis it is common practice to macerate the tissue prior to culture; this often decreases the sensitivity of culture for the zygomycetes - why? You can't really plate out a chunk of tissue from a biopsy, so the specimen is often mashed (macerated) up before preparing cultures and preparations for microscopy. The hyphae of zygomycetes are relatively non septate and essentially consist of continuous cytoplasm. In order to successfully culture a zygomycete you need to have a complete "piece" of organism bordered by the septa. Once you mash up the specimen into smaller and smaller pieces, it becomes less and less likely that you will retain a complete segment of hyphae. It is not uncommon for hyphael elements consistent with a zygomycete to be seen on direct microscopy but fail to grow in culture. With other molds, this is not a problem since septa occur regularly.

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