Mycology

(see also molecular techniques) and the Regional Mycology Laboratory Manchester

Special transport packs for collection of skin, nails or hair are available from Outfits 4752 on request.

Investigations for fungal precipitins are available; send 10mls of clotted blood.

Regional Mycology laboratory, Manchester (RMLM)

The clinical Regional Mycology Laboratory, Manchester (RMLM) is situated on the second floor of the Education & Research Centre at Wythenshawe Hospital. The RMLM provides a specialist medical mycology service for the North West of England and further afield.

Antifungal and mycological advice can be offered on the diagnosis of disease, clinical management and care of patients.

The RMLM laboratory is open from 09.00 to 17.00 hours Monday to Friday. Emergency medical advice can be obtained outwith these hours by contacting Professor Denning, Professor Richardson or the Consultant Microbiologist via the switchboard on (0161) 998 7070.

Regional Mycology laboratory, Manchester (RMLM)
Professor M D Richardson Director of RMLM	(0161) 291 2124 (laboratory) (0161) 291 5914 (Office) (0161) 291 5839 (Secretary) 07545994936 (Mobile)
Dr CB Moore, Principal Clinical Scientist	(0161) 291 5839 (office) (0161) 291 2124 (laboratory)
Fax	(0161) 291 5806
General Enquiries/Results	(0161) 291 2124
Further information is available on the Regional Mycology Laboratory web site

All samples should be sent to the Microbiology Department, Wythenshawe Hospital who will ensure appropriate transportation to the Mycology Laboratory.

Additional Tests

Additional tests can be requested by telephoning or writing to the laboratory, although it must be recognised that the archive sample available will have a limited volume.

Antifungal Drug Levels

Please ensure that details of all antifungal drugs the patient is receiving are given - this information is essential to ensure appropriate testing is performed.

Please record the date and time the specimen is taken, together with the time of last dose. These details ensure correct interpretation of results.

Indications for monitoring

• All patients receiving flucytosine

• Patients receiving itraconazole - to check drug absorption and to monitor compliance

• Patients receiving posaconazole - to check drug absorption and to monitor compliance

• All patients receiving voriconazole

• Fluconazole in patients on dialysis/haemofiltration

• Patients failing azole therapy

• If drug interactions are suspected

Specimen type	Antifungal Drug Levels
Optimal time of specimen collection	See antifungal drug table below
Correct specimen type and method of collection	Clotted blood sample (EDTA tubes are not acceptable): Adult 5ml, Neonate 0.5ml minimum
Quantity and number of specimens	See table below
Laboratory analysis	As requested
Transport	Specimens should be transported to the laboratory as soon as possible. If a delay is anticipated, samples should be refrigerated.
Turnaround time	Average turnaround of 1.4 days. All results are telephoned.
Indicative cost per sample	£55 per sample

RECOMMENDATIONS OF SAMPLING TIMES FOR ANTIFUNGAL DRUG MONITORING

NB: We do not accept EDTA tubes

Test	Pre-dose	Post-dose	Timing of Laboratory Assay Runs	Sampling Commencement and Frequency	Target Levels/Comments
Fluconazole	Just before dose*	Oral: 2-4 hours* IV: 30 minutes*	As required	Only indicated if oral dosing and short gut or a question of absorption. Discuss with laboratory.	Target concentrations have not been defined - please seek advice.
Flucytosine	Just before dose*	Oral: 2 hours* IV: 30 minutes*	As required	Commence: around second/third dose Frequency: Twice-weekly, or more often if renal function is changing	Adult: Pre-dose 30-40 mg/L Post-dose 70-80 mg/L Neonate: (<3 months) Pre-dose 20-40 mg/L Post-dose 50-80 mg/L Levels >100mg/L are potentially toxic
Itraconazole	Oral: not needed IV: just before dose	Oral: random* IV: 1hr and 4-6 hrs	Tuesdays and Thursdays Sample must be received before 2pm	Commence: Only after steady state has been reached Frequency: Dependent on patient - seek advice	A level between 5- 17mg/L is likely to be effective and non-toxic Steady state reached after 1-2 weeks on oral therapy, with little variation throughout the day
Posaconazole	Oral: not needed	Oral: random*	As required	Commence: Only after steady state has been reached Frequency: once or twice early in course and again to check compliance, or if possible drug interaction	Steady state reached after 1-2 weeks on oral therapy, with little variation throughout the day Average concentrations of >0.7 and 1.3mg/L are associated with therapeutic success for prophylaxis and treatment, respectively
Voriconazole	Just before dose* (essential for clinical management)	Not required	Monday, Wednesdays and Fridays at 12.30 by Biochemistry department, but consult Mycology Laboratory for advice	Commence: after 3 days of therapy Frequency: until therapeutic levels obtained, but repeat if a dose change, interaction or a question of poor compliance	A trough concentration between 1.3 - 5.7mg/L is likely to be effective and non-toxic. If dosage changes are required, suggest obtaining expert advice Due to the non-linear kinetics of the drug, informed clinical judgement regarding target range is not possible on any sample except pre dose samples
* these samples are most useful for clinical management

Identification and susceptibility testing of medically important fungi

Original specimens will be processed by the Microbiology Department, Wythenshawe Hospital. If fungus is isolated, the Microbiology department will then send the culture on to the Mycology Laboratory for testing. Please ensure that the request form states that mycological investigations are required.

Indications for testing

• All life threatening fungal infections, to ensure that the optimal therapy is administered

• Isolates from patients at increased risk of fungal infection, such as those infected with HIV, immunosuppressed or on ICU, so that appropriate antifungal therapy can be given

• Mucosal candidosis not responding to therapy

• Clinically significant non-Candida albicans species due to increasing incidences of both infection and fluconazole resistance

• Rare pathogens because of an increased incidence of resistance and unpredictability of resistance patterns

Specimen type	Identification and Susceptibility Testing of Yeasts and Moulds
Optimal time of specimen collection	As per Microbiology guidelines
Correct specimen type and method of collection	As per Microbiology guidelines
Quantity and number of specimens	As per Microbiology guidelines
Laboratory analysis	Full identification of all medically important yeasts and moulds. The following susceptibility tests are routinely performed :
	Yeasts Flucytosine Fluconazole Amphotericin Caspofungin Itraconazole Voriconazole	Moulds Itraconazole Amphotericin Voriconazole Posaconazole Caspofungin
	Other drugs are available upon request
Transport	As per Microbiology guidelines
Turnaround time	Identification: average turnaround of 1.8 days Susceptibility of yeasts: average turnaround of 2.2 days Susceptibility of moulds: average turnaround of 3.6 days
Indicative cost per sample	Variable depending on specimen type - contact the laboratory

Cryptococcal antigen latex agglutination test
Indications for testing: Testing for Cryptococcus neoformans capsular antigen is one of the most reliable methods for the diagnosis of cryptococcosis. Suspected cryptococcosis, including cryptococcal meningitis, pulmonary and disseminated disease, in both immunocompromised, eg. HIV-positive, and immunocompetent patients.
Cryptococcal antigen latex agglutination test	
Optimal time of specimen collection	Collection is not time dependent. When there is a suspicion of cryptococcosis
Correct specimen type and method of collection	CSF by lumbar puncture, serum, urine and BAL
Quantity and number of specimens	Minimum 500 ml CSF. Other specimen types variable. With appropriate controls, a positive test is indicative of infection. Perform repeat lumbar puncture after 2 weeks of treatment. Repeated testing can be used to monitor response to treatment, monitor for duration of treatment course, especially in HIV-positive patients.
Laboratory analysis	Latex agglutination test for cryptococcal antigen
Transport	As per Microbiology guidelines
Indicative cost per sample	£31.60

Culture and identification of dermatophytes and non-dermatophytes from skin, nail and hair

Specimen type	Culture and identification of dermatophytes and non-dermatophytes from skin, nail and hair
Optimal time of specimen collection	No specific time, when patient presents with clinical presentation of superficial fungal infection and onychomycosis
Correct specimen type and method of collection	Skin: scrapings in Dermapak, or similar envelope. Hair: plucked hair roots and hair shaft Nail: nail clippings, scrapings of sub-ungual debris Subcutaneous lesions: scrapings, punch biopsies
Quantity and number of specimens	Adequate scrapings and clippings for direct microscopy and culture
Laboratory analysis	Direct microscopy and culture for yeasts, dermatophytes and non-dermatophytes. Full identification.
Transport	As per Microbiology guidelines
Turnaround time	Direct microscopy: 1-2 days Culture and identification yeasts: 1-3 days dermatopytes: 1-3 weeks non-dermatophytes: 1 week
Indicative cost per sample	£12.54

Aspergillus galactomannan antigen ELISA

Specimen type	Aspergillus galactomannan antigen ELISA
Optimal time of specimen collection	No specific time, first clinical indication of invasive aspergillosis
Correct specimen type and method of collection	Clotted blood, serum, BAL, urine. EDTA blood is not suitable.
Quantity and number of specimens	Serum 700 l minimum or 5 ml clotted blood, prospective screening twice weekly
Laboratory analysis	Platelia ELISA for Aspergillus galactomannan circulating antigen in serum and other body fluids. Results reported as Negative, Positive (report includes cut-off value). Specificity of the test is improved if two or more consecutive specimens are positive.
Transport	As per Microbiology guidelines
Turnaround time	1-2 days
Indicative cost per sample	£46

Counterimmunoelectrophoresis for Aspergillus precipitins

Specimen type	Counterimmunoelectrophoresis for Aspergillus precipitins
Optimal time of specimen collection	No specific time, when patient presents with aspergilloma (mycetoma, fungal ball), allergic bronchopulmonary aspergillosis or other pulmonary manifestations of aspergillosis
Correct specimen type and method of collection	Clotted blood
Quantity and number of specimens	5 ml, serial samples during treatment
Laboratory analysis	CIE for Aspergillus precipitins against Aspergillus fumigatus, A. niger, A. flavus, A. terreus
Transport	As per Microbiology guidelines
Turnaround time	1-2 days
Indicative cost per sample	Screening: Aspergillus fumigatus: £14 A. fumigatus, A. flavus, A. nidulans, A. niger and A. terreus: £19 Titre: Aspergillus fumigatus: £17 A. fumigatus, A. flavus, A. nidulans, A. niger and A. terreus: £27

Surveillance of hospital environments for Aspergillus species

Specimen type	Sampling and culture for Aspergillus and other filamentous fungi in hospital environments
Optimal time of specimen collection	No specific time. Surveillance during hospital construction, maintenance, demolition and renovation, water damage, faulty air filtration and conditioning, and outbreaks
Correct specimen type and method of collection	Air samples, settled dust, surface samples, material samples
Quantity and number of specimens	Minimum: one air sample and one dust sample from patients' rooms and general hospital areas. More intensive sampling where reservoir of Aspergillus is most likely to occur.
Laboratory analysis	Culture of air samples, culture of dust and material samples
Transport	Culture plates: as per Microbiology guidelines On-site sampling, air sampling plates and material samples transported directly to RMLM
Turnaround time	5 days
Indicative cost per sample	Sampling by RMLM staff: £10.90 per hour. Culture and identification: £18.21 per sample plate.

Molecular identification of fungi from culture negative, microscopy positive specimens

Using sophisticated DNA extraction technology, fungal DNA can be obtained from most samples in which fungal hyphae are seen, including fixed paraffin sections. In some cases no sample was submitted for culture, in other cases, culture is negative.

Cases should be discussed with the RMLM staff, who will advise.

Indications for testing

Clinically significant fungal infection, with negative culture and serology.

Specimen type

Original specimen, if a fluid specimen, such as pus, BAL, pleural fluid, peritoneal fluid, transported at room temperature and stored at 4°C, -20°C or -80°C.

Fixed paraffin section (5-10 normal or thick sections placed together in a sterile container and transported at room temperature)

Turnaround time

Two weeks. Sometimes DNA extraction fails and no result is possible.