Mycology Regional Reference Unit
University Hospital of Wales
Heath Park
Cardiff
CF14 4XW
A UKAS Accredited Medical Laboratory No 9510
Public Health Wales Microbiology Cardiff
DX 6070100
Cardiff 90 CF
Tel: 029 2074 4515
Out of hours: 029 2074 7747
Fax: 029 2074 6403
| Consultant Clinical Scientist/ Head of Unit | Dr Lewis White |
| Consultant Microbiologist/Clinical Lead | Dr Matthijs Backx |
| Senior Biomedical Scientist | Lorna Vale |
COVID Statement
Patients with COVID-19 or other respiratory viral infections requiring critical care management are at increased risk of invasive fungal diseases (including invasive aspergillosis, invasive candidosis and mucormycosis). Please follow the link for guidance on the diagnosis of invasive fungal disease in critical care patients.
Invasive Fungal Disease
Invasive fungal disease is a significant complication of certain high risk haematology patients. Recommended guidance for the management if invasive fungal disease in haematology patients is provided via the link below:
Investigation and Management of Invasive Fungal Infection in Haematology Patients
Listed below are the routine services offered by the Mycology Regional Reference Unit, Cardiff.
All specimens must be accompanied with a completed Mycology Regional Reference Unit Request Form
A price list of services is currently being reviewed please contact the Mycology Regional Reference Unit for more information.
Supporting information and interpretative guidance for the tests offered by the Regional Mycology Reference Laboratory
Service |
Additional Information |
Interpretation |
Estimated Turnaround times (TAT) and sample requirements |
| Clinical advice and interpretation service | Please contact Dr Lewis White or Dr Matthijs Backx via the Mycology Reference laboratory number or hyperlinked email | N/A | N/A |
| Dermatology sample processing | Performed on skin/hair/nail samples | N/A | Up to 28 days (Please note: delays can occur, dependent on recovery and necessity to purity plate). Sample required: Hair/skin/nail sample in a sterile universal container or Dermapak. When sending hair samples please ensure the root is present as the infected area is usually close to the scalp. Pleases ensure skin scrapings are taken form the edge of skin lesions. Skin stripping’s are a poor quality specimen for fungal investigation and will be rejected. |
| Common dermatophyte identification | Please note: any isolates requesting identification that require additional purity plating in order to attain identification this will likely result in a delay in specimen turn-around time | N/A | Up to 21 days (Please note: delays can occur, dependent on recovery and necessity to purity plate). Sample required: Fungal isolate |
| Direct Microscopy | Direct microscopy is performed on respiratory and tissue specimens using light and fluorescent microscopy with calcofluor white. | Up to 5 days (dependent on day of receipt) Samples required: Respiratory samples, fluids (not blood) or tissue biopsies in a sterile universal container. |
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| Yeast ID (including molecular) |
Please download the document Identification and sensitivity testing of yeast isolates - Practice Guide [.pdf, 193kb] for further information.
Please note: in order to perform sensitivity testing it is essential to perform/confirm yeast ID.
Please note: any isolates requesting identification that require additional purity plating in order to attain identification, or if additional tests are requested post receipt (e.g. antifungal susceptibility testing) this will likely result in a delay in specimen turn-around time
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N/A | Up to 14 days (Please note: delays can occur, dependent on recovery and necessity to purity plate). Sample required: Pure yeast culture grown on a media plate or slope or a swab of the pure isolate using a sterile transport swab. |
| Yeast ID and susceptibility |
Yeast susceptibility is performed for the following antifungals:-
Results will be reported as Resistant (R), Intermediate (I) and Sensitive (S), according to MIC and international guidelines where available. Currently the method is in line with CLSI guidelines. |
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| Antifungal levels |
The monitoring of antifungal serum trough levels is reasonable in patients with:
This will permit drug dosage to be adjusted, or if this not possible to change antifungal therapy.
Assays available:
Flucytosine assay
Isavuconazole assay Itraconazole assay
Voriconazole assay
Fluconazole assay
Posaconazole assay
Please note: these tests will be performed as a send-away test by the PHE Mycology Reference Lab Bristol.
|
Voriconazole IV or Oral administration. Samples taken Pre-dose and after 3-5 days of treatment. Expected ranges: 1.0-5.5 mg/L
Itraconazole
IV or Oral administration. Samples taken Pre-dose and after 7 days of treatment.
Above 0.5 mg/L
Flucytosine
Oral administration. Samples taken Pre-dose and 2 hours post dose. IV administration. Samples taken Pre-dose and 0.5 hours post dose Therapeutic serum range: 25 to 100 mg/L
Posaconazole
Oral administration. Samples taken Pre-dose and after 5 days of treatment
Expected ranges:
Above 0.7 mg/L for prophylaxis Above 1.25 mg/L for treatment
Fluconazole
Oral administration samples taken 2 hours post dose. Expected range: Isavuconazole Expected range: |
Currently a send-away test.
Azole levels may be affected by the gel plug in a serum separation tube (SST), therefore please send sample in a plain clotted tube with no additives. |
| Candida antigen (mannan) |
Commercially available ELISA kits for the detection of mannan and antimannan antibodies are available for diagnosis of candidal infection when testing serum samples only.
Clinical evaluation is limited. The test may be useful in suspected cases of candidosis in critically ill patients and in the diagnosis of hepatosplenic candidosis.
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Both tests are best performed in combination, where the combination of mannan antigen and antibody testing was superior to either antigen or antibody in isolation. Sensitivity and specificity of the combined test was 84% and 86% respectively compared with 58% and 93% for antigen and 59% and 83% for antibody testing alone.
The presence of a positive Candida antibody result may represent Candida colonisation.
All results should be interpreted as clinically indicated along with other biomarker assays (1-3)-β-D-Glucan and Candida PCR).
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Currently send-away tests.
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| Candida antibodies (antimannan) | |||
| Aspergillus antigen (galactomannan) |
The BioRad Aspergillus Antigen assay is best performed as a screening test (1-2 times weekly), alongside the Aspergillus PCR test in patients at high-risk of developing acute invasive aspergillosis (e.g. immunosuppressed patients such as haematology patients). In this patient cohort a clotted blood (serum) sample should be sent to the Mycology Reference Laboratory.
This assay can also be used to confirm a clinical suspicion of aspergillosis (not allergic disease) in high risk patients (e.g. patients with chest radiology typical of invasive aspergillosis). In this patient a targeted respiratory specimen, such as a BAL or NBL fluid is preferential to a single blood based specimen.
Please note: Most data on performance come from high risk adult neutropenic and Stem-cell transplant patients . Utility in other patient groups (paediatrics, critical care, solid organ cancer/transplant) are less well understood
|
Please download the document Aspergillus -Indication for use of molecular diagnostics and interpretation of results [.pdf, 105kb] for further information. Please note: The performance of Aspergillus antigen (galactomannan) when testing blood is limited outside the haematology cohort. When aspergillosis is suspected in non-haematology patients a negative result should not be used to exclude disease. In these patients Aspergillus antigen testing of specimens from the focus of infection (BAL/NBL) along with (1-3)-β-D-Glucan testing of serum is recommended. If the patient is not immunosuppressed Aspergillus IgG testing can be performed. |
Up to 5 days (dependent on day of receipt). Sample requirement: |
| Aspergillus IgG antibody | The BioRad Aspergillus IgG antibody is performed on serum samples from patients at risk of chronic aspergillosis, including allergic bronchopulmonary aspergillosis, in particular cystic fibrosis patients, those with COPD or other chronic chest conditions.It is best performed alongside total and Aspergillus specifc IgE (currently performed by Immunology) that helps differentiate allergic from infective manifestations | The presence of IgG antibodies to Aspergillus may represent chronic infection, but may also be present due to allergic syndromes or colonization.
The result should be interpreted along with total and Aspergillus specific IgE and chest radiology. ABPA is best diagnosed by the presence of Total IgE (>500kuA/ml or a doubling over baseline) supported with positive Aspergillus specific serology (IgE and/or IgG) and eosiniophilia. If IgG is positive but IgE is negative then Allergic syndromes such as ABPA can be excluded and this may be indicative of chronic infection.
|
Up to 10 days (dependent on day of receipt). |
| Avian precipitation test |
Bird-fancier’s lung (Extrinsic Allergic Alveolitis) is a subset of hypersensitivity pneumonitis caused by the exposure to avian proteins present in the dry dust of the droppings and sometimes in the feathers of a variety of birds.
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Although avian preciptins is not a mycological test it is carried out at the UK Mycology reference laboratory and the send-away process is carried out by the PHW Mycology reference laboratory.
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Currently a send-away test. 5-10 ml clotted blood |
| Histoplasma serology | This test is designed to detect the presence of histoplasmosis polysaccharide antigens (HPA) in the serum of patients infected with Histoplasma capsulatum, which is endemic in parts of the USA, Central and Southern America, Africa, Asia and some European countries. | The tests should only be requested after discussion with a consultant microbiologist. Please note: These are Hazard group 3 pathogens and are dimorphic (can present as a yeast or a mold). If a patient is suspected to be at risk from these infections any fungi cultured from the patient should be handled appropriately, and travel history to endemic areas should be considered in relation to all samples received. |
Currently a send-away test. 5-10 ml clotted blood |
| Coccidioides serology | Serological diagnosis of coccidioidomycosis, which is endemic to arid/desert regions of the USA, Central and Southern America. | Currently a send-away test. 5-10 ml clotted blood |
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| Blastomyces serology | Serological diagnosis of blastomycosis, which is endemic to parts of the USA and Canada. | Currently a send-away test. 5-10 ml clotted blood |
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| Paracoccidioides serology | Serological diagnosis of paracoccidioidomycosis, which is endemic countries in Central and Southern America. | Currently a send-away test. 5-10 ml clotted blood |
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| Farmers lung antibodies |
The test detects antibodies to Saccharopolyspora retivirgula: (Formally Faenia rectivirgula) a bacterial gram-positive rod. Inhalation of the bacteria can cause the disease farmer's lung, a type of hypersensitivity pneumonitis. Handling hay bales increases exposure to the bacteria and increases the risk of developing the disease. |
Although it is a bacterial infection the testing is carried out at the UK Mycology reference laboratory and the send-away process is carried out by the PHW Mycology reference laboratory. | Currently a send-away test. 5-10 ml clotted blood |
| β-D-Glucan testing | β-D-Glucan is a major cell-wall component of various medically important fungi, except mucorales species where it is not present and Cryptococcus species where it is present in limited concentrations.
It is a protease zymogen-based colorimetric assay for the qualitative detection of (1-3)-β-D-Glucan in the serum/plasma of patients with symptoms of, or medical conditions predisposing the patient to, invasive fungal disease.
Please note: Haemolysed blood cannot be tested.
Please note: Testing respiratory specimens for the presence of β-D-Glucan is not recommended.
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The concentration of (1-3)-β-D-Glucan in serum/plasma samples can be used as an aid in the diagnosis of deep-seated mycoses and fungaemias but a positive result does not indicate which genera of fungi may be causing infection and although cut-offs are available to determine positivity (≥80pg/ml) they were defined for the diagnosis of invasive candidal disease and there have been no attempts to re-optimise for individual fungal pathogens other than Candida.
The specificity of the assay is improved (99%) if two consecutive positive results are attained, and a single result could represent false positivity.
|
1-3 days Sample required 5-10 ml Clotted sample |
| Cryptococcus antigen |
The IMMY Cryptococcal lateral flow assay is performed on serum and CSF samples. It should be performed primarily on immuno-suppressed individuals at risk and with symptoms of cryptococcal meningitis (e.g HIV), although recently cases of cryptococcosis cause by C. gattii have been documented in immuno-competent hosts.
The assay shows excellent agreement (98.5%) and correlation (r2: 0.93) with previously used assays (CRAG LA). The LFA has lower limit of detection for all four serotypes of Cryptococcus neoformans (A-D), compared to the alternatives. Recently, the assay has been proposed as the new gold standard for the diagnosis of cryptococcal disease.
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Antigen testing on serum and CSF has established clinical utility for Cryptococcal disease and can be used not only to diagnose disease but also to provide prognostic information and monitor response to treatment.
A negative test excludes disease and positive tests with a titre >1/16 have a high PPV.
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1-3 days (dependent on day of receipt). Sample requirement 1-5 ml Clotted blood At least 0.5 ml or more of CSF in a sterile universal container. |
| Mould ID Phenotypic |
Please note: in order to perform sensitivity testing it is essential to perform/confirm mould ID.
Please note: any isolates requesting identification that require additional purity plating in order to attain identification, or if additional tests are requested post receipt (e.g. antifungal susceptibility testing) will likely to result to a delay in specimen turn-around time
|
N/A |
Up to 14 days (Please note: delays can occur, dependent on recovery and necessity to purity plate). Sample requirement: A pure growth of the target organism on a Sab Agar Slope. |
| Mould ID Molecular | Please note: due to limited demand these tests will be performed as a send-away test by the PHE Mycology Reference Lab Bristol | N/A | Currently a send-away test. |
| PCR Aspergillus | Aspergillus PCR testing is best performed as a screening test (1-2 times a week), alongside Aspergillus antigen testing of patients who are high-risk of developing acute invasive aspergillosis (e.g. immunosuppressed patients such as haematology patients). In this patient cohort, an EDTA (plasma) sample should be sent to the Mycology Reference Unit. This assay can also be used to confirm a clinical suspicion of aspergillosis (not allergic disease) in high risk patients (e.g. patients with chest radiology typical of invasive aspergillosis). In this patient a targeted respiratory specimen, such as a BAL or NBL fluid is preferential to a single blood based specimen. | Please download the document Aspergillus -Indication for use of molecular diagnostics and interpretation of results [.pdf, 105kb] for further information |
Up to 5 days (dependent on day of receipt). Sample requirement |
| PCR - A. fumigatus resistance | By using the commercially available Pathonostics Aspergenius assay the most common mutations (TR34/L98H and TR46/Y121F/T289A) associated with azole resistance in A. fumigatus can be determined. This can be performed on A. fumigatus isolates or direct from plasma/serum or BAL/NBL samples, provided that there is sufficient fungal burden within the clinical specimen | As the international guidelines for the management of invasive apergillosis recommend voriconaozle as front-line therapy detecting the presence of mutations that potential infer azole resistance allows the management of cases of aspergillosis to be adjusted accordingly.
The TR34/L98H mutation induces pan-azole resistance, whereas the TR46/Y121F/T289A mutation induces high-level resistance to voriconazole and variable resistance to the other azoles.
The assay also has the capacity to identify cases caused by A. terreus which may be resistant to amphotericin B.
The mutation assay has been shown to be 100% specific to their target sequences. |
Up to 5 days (dependent on day of receipt). Please note: Resistance PCR will only be performed when a sample is strongly positive by an initial Aspergillus PCR test. |
| PCR Pneumocystis jiroveci |
Pneumocystis PCR is more sensitive than immunofluoresence and can be performed on respiratory specimens including bronchoalveolar lavage and NBL. The assay has also been validated for the testing of nasopharyngeal swabs although sensitivity is reduced (see interpretation).
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When testing deeper respiratory samples (BAL, NBL) Pneumocystis PCR is highly sensitive (>95%) and consequently, if negative is a good indicator to exclude PCP. When interpreting Pneumocystis PCR positive results from deeper respiratory samples the patient’s underlying clinical condition should be considered when deciding on the clinical significance of the result. For HIV positive patients strong PCP positive results would be expected (Ct: <30 cycles), whereas in haematology and SOT recipients moderate values (Ct: 30-35 cycles) should also be considered significant. PCP positives >35 cycles should be interpreted as clinically indicated and could represent colonisation, although in patients capable of raising a substantial host response it is important to consider the influence of host immunity on fungal burden and the patient’s symptoms.
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Up to 5 days (dependent on day of receipt). Sample requirements |
| PCR Candida |
Candida PCR can be performed on serum/plasma and fresh tissue samples. The testing of respiratory samples is not recommended. It is best used alongside(1-3)-β-D-Glucan testing, although it can be used to complement Candida antigen and antibody testing
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It has been evaluated and standardized on a UK wide basis. PCR positivity rates of 85% in patients with proven or probable IC were 85% compare with 38% for blood cultures. As the specificity of the local Candida PCR is 97% it is useful for confirming a diagnosis of invasive candidal disease, although it should not be used to exclude IC. |
Up to 7 days (dependent on day of receipt). Candida PCR is not currently ISO 15189 accredited |
| PCR - Tissue | PCR can be performed on tissue but limits of sensitivity and specificity are not established. Formalin fixed tissue requires extensive manipulation and degradation of nucleic acid means that false negative results can occur. Currently, only fresh tissue is tested locally and formalin fixed material will be sent to PHE Mycology Reference Lab Bristol |
7-10 days* Sample requirement fresh tissue Tissue PCR is not currently ISO 15189 accredited |
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| PCR Pan Fungal | Detection of fungi in clinical specimens using PCR. If PCR is positive identification may be possible through sequencing of the amplicon. | Given the pan-fungal approach false positivity may occur and results should always be interpreted as clinically indicated. Combining Pan-fungal PCR with (1-3)-β-D-Glucan testing may improve clinical accuracy | 7 -10 days* Sample requirement 5-10 ml EDTA/Clotted blood*. Fresh tissue in a sterile universal container. At least 0.5 ml of CSF in a sterile universal container. The testing of respiratory samples is not recommended. *This may be performed as a send-away test dependant on restraints on the laboratory at time of receipt. Pan-fungal PCR is not currently ISO 15189 accredited |
| SNP Analysis | he presence of single nucleotide polymorphisms (SNPs) in the genes for Dectin 1 and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) can pre-dispose patients to invasive aspergillosis. Currently, only available as an assay under evaluation it should be performed as part of the haematology screening strategy for IA and performed pre and post SCT to determine potential risk. If multiple SNPs are present and the patient is Aspergillus PCR positive then risk of IA is >50%. | 7-10 days* Sample requirement 5-10 ml EDTA/Clotted blood*. Fresh tissue in a sterile universal container. SNP analysis is not currently ISO 15189 accredited |
All specimens must be accompanied with a completed Mycology Regional Reference Unit Request Form
A price list of services is currently being reviewed please contact the Mycology Regional Reference Unit for more information.
For consideration
Assay |
Additional Information |
| PCR - Tissue |
PCR can be performed on tissue but limits of sensitivity and specificity are not established. Formalin fixed tissue requires extensive manipulation and degradation of nucleic acid means that false negative results can occur.
Currently, only fresh tissue is tested locally and formalin fixed material will be sent to PHE Mycology Reference Lab Bristol |
| PCR Pan fungal |
This is performed as a send-away assay by PHE Mycology Reference Lab Bristol.
Given the pan-fungal approach false positivity may occur and results should always be interpreted as clinically indicated. Combining Pan-fungal PCR with b-D-Glucan testing may improve clinical accuracy |
| SNP Analysis | The presence of single nucleotide polymorphisms (SNPs) in the genes for Dectin 1 and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) can pre-dispose patients to invasive aspergillosis. Currently, only available as an assay under evaluation it should be performed as part of the haematology screening strategy for IA and performed pre and post SCT to determine potential risk. If multiple SNPs are present and the patient is Aspergillus PCR positive then risk of IA is >50%. |
Tools and Links
Defining and managing COVID-19-associated pulmonary aspergillosis: the 2020 ECMM/ISHAM consensus criteria for research and clinical guidance.
Philipp Koehler, Matteo Bassetti, Arunaloke Chakrabarti, Sharon C A Chen, Arnaldo Lopes Colombo, Martin Hoenigl, Nikolay Klimko, Cornelia Lass-Flörl, Rita O Oladele, Donald C Vinh, Li-Ping Zhu, Boris Böll, Roger Brüggemann, Jean-Pierre Gangneux, John R Perfect, Thomas F Patterson, Thorsten Persigehl, Jacques F Meis, Luis Ostrosky-Zeichner, P Lewis White, Paul E Verweij, Oliver A Cornely,
The Lancet Infectious Diseases,
Volume 21, Issue 6,
2021,
Pages e149-e162,
ISSN 1473-3099,
https://doi.org/10.1016/S1473-3099(20)30847-1
Pulmonary Aspergillosis in Patients with Suspected Ventilator-associated Pneumonia in UK ICUs.
Loughlin L, Hellyer TP, White PL, McAuley DF, Conway Morris A, Posso RB, Richardson MD, Denning DW, Simpson AJ, McMullan R.
American Journal of Respiratory and Critical Care Med. 2020 Oct 15;202(8):1125-1132.https://doi.org/10.1164/rccm.202002-0355OC .
Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium.
J Peter Donnelly, Sharon C Chen, Carol A Kauffman, William J Steinbach, John W Baddley, Paul E Verweij, Cornelius J Clancy, John R Wingard, Shawn R Lockhart, Andreas H Groll, Tania C Sorrell, Matteo Bassetti, Hamdi Akan, Barbara D Alexander, David Andes, Elie Azoulay, Ralf Bialek, Robert W Bradsher, Jr, Stephane Bretagne, Thierry Calandra, Angela M Caliendo, Elio Castagnola, Mario Cruciani, Manuel Cuenca-Estrella, Catherine F Decker, Sujal R Desai, Brian Fisher, Thomas Harrison, Claus Peter Heussel, Henrik E Jensen, Christopher C Kibbler, Dimitrios P Kontoyiannis, Bart-Jan Kullberg, Katrien Lagrou, Frédéric Lamoth, Thomas Lehrnbecher, Jurgen Loeffler, Olivier Lortholary, Johan Maertens, Oscar Marchetti, Kieren A Marr, Henry Masur, Jacques F Meis, C Orla Morrisey, Marcio Nucci, Luis Ostrosky-Zeichner, Livio Pagano, Thomas F Patterson, John R Perfect, Zdenek Racil, Emmanuel Roilides, Marcus Ruhnke, Cornelia Schaefer Prokop, Shmuel Shoham, Monica A Slavin, David A Stevens, George R Thompson, III, Jose A Vazquez, Claudio Viscoli, Thomas J Walsh, Adilia Warris, L Joseph Wheat, P Lewis White, Theoklis E Zaoutis, Peter G Pappas,
Clinical Infectious Diseases, Volume 71, Issue 6, 15 September 2020, Pages 1367–1376, https://doi.org/10.1093/cid/ciz1008
A National Strategy to Diagnose Coronavirus Disease 2019–Associated Invasive Fungal Disease in the Intensive Care Unit.
P Lewis White, Rishi Dhillon, Alan Cordey, Harriet Hughes, Federica Faggian, Shuchita Soni, Manish Pandey, Harriet Whitaker, Alex May, Matt Morgan, Matthew P Wise, Brendan Healy, Ian Blyth, Jessica S Price, Lorna Vale, Raquel Posso, Joanna Kronda, Adam Blackwood, Hannah Rafferty, Amy Moffitt, Alexandra Tsitsopoulou, Soma Gaur, Tom Holmes, Matthijs Backx,
Clinical Infectious Diseases, 2020;, ciaa1298, https://doi.org/10.1093/cid/ciaa1298
White PL, Wiederhold NP, Loeffler J, Najvar LK, Melchers W, Herrera M, Bretagne S, Wickes B, Kirkpatrick WR, Barnes RA, Donnelly JP, Patterson TF. 2016. Comparison of nonculture blood-based tests for diagnosing invasive aspergillosis in an animal model. J Clin Microbiol 54:960–966. doi:10.1128/JCM.03233-15. [click here to access full article]
Springer J, White PL, Hamilton S, Michel D, Barnes RA, Einsele H, Löffler J. 2016. Comparison of performance characteristics ofAspergillus PCR in testing a range of blood-based samples in accordance with international methodological recommendations. J Clin Microbiol 54:705–711. doi:10.1128/JCM.02814-15. [click here to access full article]
White PL, Barnes RA, Springer J, Klingspor L, Cuenca-Estrella M, Morton CO, Lagrou K, Bretagne S, Melchers WJG, Mengoli C, Donnelly JP, Heinz WJ, Loeffler J, the EAPCRI. 2015. Clinical performance of Aspergillus PCR for testing serum and plasma: a study by the European Aspergillus PCR Initiative. J Clin Microbiol 53:2832–2837. doi:10.1128/JCM.00905-15. [click here to access full article]