Mycology

It is important that good quality samples are sent to the laboratory otherwise results may be misleading and repeat specimens indicated.

Skin and Nail Specimens should be collected into a Dermapak or similar black card packet. If these are not available then a sterile, plain, plastic universal may be used. Please do not use ad-hoc packets or sellotape as culture may fail due to overgrowth of contaminating saprophytic fungi. Microscopy will be impeded by specimen affixed to sellotape. If toothbrushes are used to sample scalp skin, please note this prevents microscopic examination of the sample. A scalp skin scraping or hair pluck is preferable.

Sample collection

Clean lesions with surgical spirit or 70% alcohol before collecting samples. This minimizes contamination and is an aid to microscopy if greasy ointments or powders have been applied

Be sure to include as much material as possible so that full laboratory investigations can be carried out. It is always useful to have enough skin or nail to repeat the culture if necessary

• Skin : Collect material by scraping from the edges of the lesions, ideally with a scalpel blade.
• Nail : If possible, collect the subungual debris in addition to nail clippings. Sample the discolored, dystrophic or brittle parts of the nail only, sampling as far back as possible from the distal part of the nail.
• Hair : Pluck hairs from the affected area with forceps (infected hairs come out easily) and scrape the scalp with a scalpel. Preferably, the sample should include hair roots, the contents of plugged follicles and skin scales. Hair cut with a scissors is unsatisfactory as the focus of infection is usually below or near the surface of the scalp. Sterile toothbrushes may be useful but preclude microscopy on the sample.

Storage of specimens

Skin and nail specimens should be stored at room temperature, as dermatophyte fungi may be killed at 4 to 6 ^oC. Swabs from mucous membranes should be stored in the refrigerator prior to dispatch.

Use(s):

Diagnosis of fungal infection by microscopy and culture.

Description:

Microscopy for yeasts, mycelium and other fungal elements; culture of any viable fungi present and identification of any clinically significant species. Antifungal susceptibility testing is undertaken where appropriate or requested.

Results:

Microscopy is reported as “No fungus seen” or positive with a description of the fungal cells seen.
Culture is reported as the identity of any significant fungi isolated.

Specimens other than skin, hair and nails that may require fungal culture include oral and vaginal swabs; urine; peritoneal dialysis fluid; CSF; respiratory samples (e.g. sputum, broncho-alveolar lavage fluid), tissues.  Sterile white topped universals should be used to send these samples, or smaller sterile tubes if the volume is small to avoid evaporation during transit. However if referring from other laboratories we advise processing the primary sample and then sending resultant fungal cultures for identification or further testing.

Swabs from mucous membranes: These should be in plain transport medium, as yeasts die rapidly in dry swabs and use of charcoal swabs interferes with microscopic examination.

Oral rinses are the best specimen for oral candidosis. Ask the patient to rinse 10ml sterile saline in their mouth for about 30 seconds then spit back into a sterile universal tube and send.

Use(s):

Diagnosis of fungal infection by microscopy and culture.

For most sample types (with the exception of skin, nail and hair), we would advise referring laboratories to process the primary sample and then send resultant fungal cultures for identification or further testing

Description:

Microscopy for yeasts, mycelium and other fungal elements; culture of any viable fungi present and identification of any clinically significant species. Antifungal susceptibility testing is undertaken where appropriate or requested.

Results:

Microscopy is reported as “No fungus seen” or positive with a description of the fungal cells seen.
Culture is reported as the identity of any significant fungi isolated, estimation of amount of fungal growth (+,++,+++) where relevant.

Culture of yeasts should ideally be sent on a Sabouraud’s agar slope in a bijou or universal.

Use(s):

Identification of yeasts and assessment of susceptibility to antifungals.

Description:

Identification, usually to species level using a combination of morphological and nutritional/enzymatic tests.  Molecular identification is carried out for those isolates which cannot be identified using phenotypic tests.

Susceptibility testing by CLSI M44A disc diffusion (fluconazole) or microbroth dilution (fluconazole, itraconazole, voriconazole, amphotericin B, flucytosine, caspofungin, posaconazole, anidulafungin and micafungin). Specific antifungal(s) tested depend on the identity and source of the isolate and the clinical details supplied. Microbroth dilution testing is undertaken where indicated by isolate identity, disc diffusion results, or where requested specifically. The identity of the yeast isolate is always confirmed or carried out on isolates sent for sensitivity testing.

Results:

Identity of the yeast, usually to species level, sensitivity to one or more antifungal agents

Susceptibility testing results, Clinical breakpoints (CBP) and Epidemiological cutoff values (ECV)

When an antifungal is reported as being “Susceptible” it means that it is likely the patient will respond to this antifungal at standard doses.

When an antifungal is reported as being “Susceptible dose-dependant” or “Intermediate” it means it is likely the patient will respond to this antifungal at higher than standard doses.

When an antifungal is reported as being “resistant”, it means the patient is unlikely to respond to this antifungal.

Clinical breakpoints are defined as the highest minimum inhibitory concentration (MIC) at which a full response can normally be expected (in typical cases of infection with therapeutic drug levels) and is based on outcome data in clinical trials and other studies. There are relatively few fungus/antifungal combinations for which this data is available. Susceptible-dose dependant or intermediate indicates response can normally be expected at a higher than standard dose. Resistance indicates that there is a relatively high likelihood that a patient will not have a positive outcome on that antifungal and it is usually advisable to avoid using it.

Epidemiological cutoff values are the highest MIC seen in wild type (WT) populations of the species that have not been exposed to the antifungal, and where the MIC is above the WT, it is considered Non-wild type and indicates that there is an increased likelihood that a fungus will not be inhibited by the antifungal in question and that an infection will not respond to that antifungal. However, there is no evidence available in terms of clinical outcomes on which to base a CBP.

For both CBPs and ECV it is important to state that outcomes from  any fungal infection treated with any antifungal will be a function of the fungal phenotype, the pharmokinetics of the antifungal in the patient being treated and the response of the patient’s immune systems. CBPs and ECVS simply provide useful guidance in choosing a therapeutic option.

The CBPs and ECVs for our asays are based on the Clinical Laboratory Standards Institute (CLSI) methodology or assays validated by this methodology.

Where there are CBPs susceptibility results are reported as “susceptible”, “susceptible dose dependant”, “intermediate” or “resistant”.  Where there are ECVs, wild type , results are reported as “susceptible”, and non-wild type results are reported as “resistant”. MICs are available for some isolates on request.

Please feel free to contact mycology to discuss any sensitivity results.

Culture of mould should ideally be sent on a Sabouraud’s agar slope in a bijou or universal.

Use(s):

Identification of moulds.

Description:

Identification, usually to species level on the basis of macroscopic and microscopic morphology. Molecular identification is carried out for those isolates which cannot be identified using phenotypic tests.

Specimens:

Culture of mould, ideally on a Sabouraud’s agar slope in a bijou or universal.

Results:

Identity of the mould, usually to species level.

Note:

Susceptibility testing of mould isolates can be carried out on request.

Identification of Environmental Fungi

Identification of yeasts and moulds from environmental sources can be carried out after discussing your requirement with the laboratory. Please contact Dr Richard Barton to discuss.

Note:

Culture of environmental specimens is carried out by prior arrangement and on medical or environmental health referral only. Costs are dependent on the extent and complexity of the investigations

For the following tests, please send serum (700 µl minimum) or 7 mL clotted blood in a plain tube; EDTA blood is not suitable.

BAL and CSF samples can also be tested (700 µl minimum). Send in white topped universal.

Use(s):

Diagnosis of invasive aspergillosis usually in immunocompromised patients.

Description:

Determination of the presence of Aspergillus antigen (galactomannan) in serum or BAL by ELISA.

Please note we cannot apply this test to Brochical washings or Tracheal aspirates..

Results:

Negative; Positive with the index value (indicating the relative concentration of galactomannan)

Note:

Positive results are confirmed before reporting by re-testing the specimen submitted. To improve specificity positives should be confirmed by submission of a second specimen.

The uncertainty of measurement of this assay has been characterised in the following way, a control sample near the cutoff index of 1.0, tested over 4 months (every run performed on a single batch of kits) yielded a mean of the Optical Density of 0.410 and a standard deviation of 0.05, thus we estimate that 95% of all samples at this level will have a range of  + or – 0.09 (1.96 x 0.04) in OD, which is + or – 0.18 in Index. Therefore samples with an index value of 0.230-0.49 should be considered possible false negatives and repeat tests requested. Equally samples with an index of up to 0.590 could be false positives, however all positives are retested and it is highly unlikely (< 95%) that this false positive would test positive again.

For the following tests, please send serum (300 µl minimum) or 3mL clotted blood in a plain tube; EDTA blood is not suitable.

Please note this test is provided by the Immunology Department. Clinical advice is available from the Mycology Clinical Scientists.

Use(s):

Diagnosis of allergic bronchopulmonary aspergillosis, aspergilloma, paranasal sinus aspergillosis, other forms of aspergillosis in immunocompetent patients.

Description:

Quantitation of IgG antibodies to Aspergillus fumigatus in serum using a commercial automated Fluorescent Immuno Enzyme Assay (ImmunoCAP).

Results:

Results are returned as mg Antibody per litre (mgA/L) and range from <2.0 to >200.

Interpretation:

As Aspergillus fumigatus spores are common in the environment and almost everyone is exposed to them, low levels of antibody to A. fumigatus are found in many patients and are not clinically significant. However, the level of antibody which is considered significant is dependant upon the underlying conditions of the patient. In people with Cystic Fibrosis (CF) there are relatively higher levels of antibody to A. fumigatus compared to other patient groups; for CF patients levels of antibody >90 mgA/L are considered significant. In non-CF patients levels of antibody >40 mgA/L are considered significant.

Note:

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at the approximate level the same as the cutoff for a positive result (40mg/L), tested over 8 months yielded a mean of 49.95 mg/L and a standard deviation of 3.53, thus we estimate that 95% of all samples at this level will have a range from + or – 6.92 mg/L.

For the following tests, please send serum (300 µl minimum) or 3mL clotted blood in a plain tube; EDTA blood is not suitable.

Please note this test is provided by the Immunology Department. Clinical advice is available from the Mycology Clinical Scientists

Use(s):

Detection of antibodies to pigeon serum or budgerigar antigens for diagnosis of Bird Fanciers’ Lung.

Description:

Determination of the presence and levels of IgG antibodies to pigeon serum or budgerigar antigen mix (serum, feathers, droppings). The method used is a commercial automated Fluorescent Immuno Enzyme Assay (ImmunoCAP).

Results:

Results are returned as mg Antibody per litre (mgA/L) and range from <2.0 to >200.

Interpretation:

It has been shown that IgG antibody levels to pigeon serum > 10mgA/L are clinically significant and correlate well with significant exposure to both pigeon and other bird antigens. In symptomatic patients such levels are highly likely to indicate Bird Fancier’s Lung. Similarly, it has been shown that serum IgG antibody levels to Budgie mix antignes >8mgA/L are likely to indicate sensitivity to that antigen source in symptomatic patients.

Note:

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at the approximate level the same as the cutoff for a positive result (10mg/L), tested over 8 months yielded a mean of 16.45 mg/L and a standard deviation of 1.21, thus we estimate that 95% of all samples at this level will have a range from + or – 2.38 mg/L.

For the following tests, please send serum (300 µl minimum) or 3mL clotted blood in a plain tube; EDTA blood is not suitable.

Please note this test is provided by the Immunology Department. Clinical advice is available from the Mycology Clinical Scientists.

Use(s):

Detection of antibodies to Micropolyspora faeni also known as Saccharopolyspora rectivirgul and Thermoactinomyces vulgaris to diagnose Farmers’ Lung

Description:

Determination of the presence of IgG antibodies to M. faeni and T. vulgaris in serum using a commercial automated Fluorescent Immuno Enzyme Assay (ImmunoCAP).

Results:

Concentration of IgG antibodies to M. faeni and T. vulgaris in mg/L. Positive results with IgG to either M. faeni or T. vulgaris antigens >39.9mg/L is likely to indicate that thermophilic actinomycets are the cause of the patients HP. Levels of IgG to M faeni between 36-39.9mg/L or to T. vulgaris between 22 and 39.9mg/L indicate HP may relate to actinomycete antigen exposure and further testing and/or antigen avoidance may be advised and results will be liaised.

Note

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at a similar level the same as the cutoff for a positive result (M. faeni 22mg/L, T. vulgaris 36mg/L), tested over 2 months yielded a mean of 35.6 mg/L (M. faeni) 36.3 (T. vulgaris) and we estimate that 95% of all samples at this level will have a range from + or – 4.5 mg/L (M. faeni) and 6.6 (T. vulgaris).

Out of scope of ISO 15189:2012 accreditation.

For the following tests, please send serum (200 µl minimum) or 3mL clotted blood in a plain tube; EDTA blood is not suitable.

Use(s):

Diagnosis of fungal infection, exclusion of fungal infection (if negative).

Description:

Determination of the presence of 1-3 Beta- D- glucan (fungal cell wall antigen) in serum by kinetic enzyme assay

Results:

30 to 59 pg/mL Negative, 60-80 pg/mL Indeterminate, >80pg/mL Positive.

Note:

Positives may be due to contaminating substances such as surgical gauze, IV Immunoglobulins or gut leakage. Positives may represent several different fungal disases including Candidosis, Aspergillosis and Pneumocystis and further testing is usually indicated. Samples from patient s with Cryptococcosis and Mucoromycosis are not positive for BDG.

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample near the cutoff for this test at 60pg/ml, was tested over 6 months (79 runs) and yielded a mean of 45.8 pg/mL and a standard deviation of 6.3, we estimate that 95% of all samples at this level will have a range from + or – 13.7pg/mL.

Use(s):

Confirmation of adequate levels and alerting to toxic levels in patients receiving itraconazole for treatment or prophylaxis of fungal disease.

Results:

Drug concentration in mg/L, with advice on target levels.  Levels should be maintained above 0.5 mg/L. If levels are below this: ensure dosing is appropriate in relation to meals – consider increasing dose or examine effects of concomitant medications.

Note:

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at the approximate level the same as the cutoff for a therapeutic level, ie 0.5mg/L, tested over 8 months yielded a mean of 0.74 mg/L and a standard deviation of 0.029, thus we estimate that 95% of all samples at this level will have a range from + or – 0.06 (1.96 x 0.029) mg/L.

Use(s):

Confirmation of adequate levels and alerting to toxic levels in patients receiving voriconazole for treatment or prophylaxis of fungal disease.

Results:

Drug concentration in mg/L, with advice on target levels. Trough levels should be maintained between 1 and 5.5mg/L.  If levels are below this: ensure dosing is appropriate in relation to meals (if given orally) – consider increasing dose or examining effects of concomitant medications.

Note:

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at the approximate level similar to the cutoff for a therapeutic level, ie 1.0mg/L , tested over 8 months yielded a mean 1.14mg/L and a standard deviation of 0.03, thus we estimate that 95% of all samples at this level will have a range from + or – 0.065 (1.96 x 0.03 ) mg/L.

Use(s):

Confirmation of adequate levels in patients receiving posaconazole for treatment or prophylaxis of fungal disease.

Results:

Drug concentration in mg/L, with advice on target levels. Trough levels should be maintained above 0.7mg/L for prophylaxis and above 1.25mg/L for treatment. If levels are below this – ensure drug is taken with fatty food, consider increasing dose (max 800mg/d) or examining concomitant medications.

Note:

The uncertainty of measurement of this assay has been characterised in the following way, a positive control sample at the approximate level a similar level as the cutoff for a prophylactic level, ie 0.7mg/L , tested over 8 months yielded a mean of 1.06 mg/L and a standard deviation of 0.035, thus we estimate that 95% of all samples at this level will have a range from + or – 0.068 (1.96 x 0.035) mg/L.