Review

British Society for Medical Mycology best practice recommendations for the diagnosis of serious fungal diseases: 2025 update

Silke Schelenz, Alireza Abdolrasouli, Darius Armstrong-James, et al

Lancet Infect Dis Available online 10 November 2025

https://doi.org/10.1016/S1473-3099(25)00550-X

Summary

The fungal diagnostic landscape is evolving; whereas previously, traditional culture-based methods dominated, most invasive fungal disease is now diagnosed with non-culture-based tests, including direct microscopy, antigen, antibody, and molecular assays, supported by histopathology and radiology. Access to and turnaround time of diagnostic tests and their clinical implementation varies across the UK. The British Society for Medical Mycology convened an expert group to update its 2015 best practice guidance. Guidance on histopathology and radiology investigations remain unchanged from the 2015 standards of care. For each mycological test, we include test-specific commentaries and accompanying tables, with expected turnaround times (sample collection to reporting). Based on recent evidence, new or stronger recommendations include use of Pneumocystis PCR; 1,3-β-D-glucan testing for suspected invasive candidiasis and Pneumocystis pneumonia; higher volume respiratory sample cultures; Aspergillus antigen or antibody-based testing in expanded clinically vulnerable populations, including patients in intensive care units and patients with chronic respiratory disease (including asthma); use of Candida PCR and Mucorales PCR in specific contexts; pan-fungal PCR and DNA sequencing for fungal identification from positive microscopy or histopathology specimens; and inclusion of posaconazole and isavuconazole in therapeutic antifungal monitoring recommendations. We discuss integration of diagnostic tests with antifungal stewardship and common clinical scenarios. Although recommendations focus on adults and UK practice, use in paediatric populations and worldwide applicability is discussed. Recommendations are presented as auditable standards to facilitate implementation and quality improvement measures. An emphasis on integration of combined diagnostics with antifungal stewardship and clinical pathways extends guideline relevance beyond microbiology laboratories to clinicians investigating patients with multimorbidity and suspected fungal disease within increasingly complex health-care systems.

Panel

Microbiology best practice recommendations

Clinical requests

All test requests should state whether and how the patient is immunocompromised, as per 2015 best practice²

Reporting to clinicians

All key positive results (including positive blood cultures, positive microscopy on deep specimens, and positive antigen or molecular tests) should be actively communicated to clinical staff within 2 h

Microbiology

Specimen collection and processing

• •Samples should preferably be collected from the infected site before antifungal therapy is started
• •Laboratory should apply mycology-specific methods for processing samples
  • •Consider spinning fluids and do not grind tissues

Direct microscopic examination

• •Direct microscopy is recommended for all tissues and fluids from normally sterile sites using an adequate stain
  • •The use of optical brighteners (eg, Calcofluor–White) should be considered for rapid direct microscopy
• •For the diagnosis of cryptococcal meningitis in cerebrospinal fluid, antigen testing can substitute for India ink microscopy, if done rapidly
• •Direct microscopy from non-sterile sites (eg, bronchoalveolar lavage fluid [BALF]) is recommended where patients are being investigated for invasive fungal infections
• •Direct microscopy for fungi on samples from normally sterile sites should be available within 2–4 h from arrival in the laboratory

Fungal culture and identification, storage of isolates

• •Mycology-specific, standardised operative procedures are recommended for the culture of fungi
• •To enhance the sensitivity for the detection of fungi in blood cultures, a minimum of 60 mL blood (adults) should be obtained
• •High volume culture of respiratory samples (>100 μL) should be applied on all patients at risk of fungal infection
• •All fungi (yeasts and moulds) isolated from sterile sites and moulds from the lung should be identified to the species level
  • •If Cryptococcus is suspected, yeasts should also be identified from respiratory samples
• •Yeasts cultured from urine specimens from patients at high risk of infection (in critical care or immunocompromised) should be identified to genus or species level
• •Fungal cultures should be reported by the genus and species (common yeasts) or species or complex name (rare yeasts and moulds)
• •Yeasts from normally sterile sites or those that exhibit an unusual resistance profile should be stored for 3 months
• •Mould cultures should be kept until full identification and antifungal susceptibility testing (when applicable) are completed or 3–6 months
  • •In suspected outbreak, yeast or mould isolates are advised to be stored for at least 3 months

Susceptibility testing

• •Antifungal susceptibility testing should be routinely performed on all fungal isolates suspected of being the cause of a severe, deep-seated, or life-threatening invasive infection
• •Testing should also be done if the fungal infection is refractory to treatment or a breakthrough infection
• •Species level identification followed by susceptibility testing is recommended if antifungal therapy is being administered

Fungal biomarkers and antigens

• •Test for cryptococcal antigen on all cerebrospinal fluid specimens from patient groups at high risk of infection
• •Serum galactomannan should be performed whenever invasive aspergillosis is suspected, notably among patients in medical intensive care and those who are immunocompromised
  • •Active galactomannan surveillance in new groups at risk (eg, people with influenza, COVID-19, or interstitial lung disease) is required as clinical and radiological features of invasive aspergillosis are non-descript
  • •Although serum galactomannan in patients without neutropenia might have reduced sensitivity, specificity remains good
• •BALF galactomannan should be performed in the same groups as for serum, and in patients with complex respiratory conditions with an uncertain diagnosis
• •Serum 1,3-β-d-glucan (BDG) should be performed in patients with suspected of invasive candidiasis or Pneumocystis pneumonia
  • •It might be of value in supporting a diagnosis of invasive aspergillosis
  • •Serum BDG provides good sensitivity for the diagnosis of Pneumocystis pneumonia
  • •The negative predictive value of BDG is typically sufficient to exclude invasive yeast and Pneumocystis, but positivity should be combined with additional mycological testing

Serology (fungal specific IgE, IgG)

• •All individuals newly diagnosed with asthma should be screened for A fumigatus sensitisation using a fungus-specific IgE or allergy skin testing including Aspergillus
• •Patients with one or more pulmonary cavities should be tested for Aspergillus IgG
  • •Aspergillus IgG is the key assay for chronic pulmonary aspergillosis and might be positive in invasive aspergillosis, especially in patients without neutropenia

Molecular diagnostics

• •Pneumocystis PCR should be done in BALF from all patients who are immunocompromised and might also be useful in (induced) sputum and endotracheal and upper respiratory tract specimens
  • •Nasopharyngeal PCR specimens in young children with possible Pneumocystis pneumonia are usually the only means of establishing the diagnosis
• •Combining serum BDG and Pneumocystis PCR is useful for both confirming and excluding Pneumocystis pneumonia
• •Aspergillus PCR should be performed on respiratory (particularly deep) samples where any form of aspergillosis is suspected
  • •Aspergillus PCR testing of blood can be considered, alongside galactomannan in patients at high risk of invasive aspergillosis (eg, patients with neutropenia)
  • •A positive Aspergillus PCR combined with positive galactomannan usually confirms the diagnosis of invasive aspergillosis, and if both negative, rules it out
• •Mucorales PCR should be considered in patients at high risk where there is clinical evidence of invasive fungal disease, particularly if other causes (eg, aspergillosis) have been excluded
  • •Dual Mucorales and Aspergillus infections are increasingly documented in patients with neutropenic and patients with rhinosinusitis
  • •Mucorales PCR on BALF can both confirm and exclude pulmonary disease, whereas PCR on blood and tissue can confirm, but not necessarily exclude disease
• •Where available, Candida PCR should be combined with serum BDG for testing of patients at high risk of invasive disease (eg, patients in intensive care units with several risk factors)
• •Pan-fungal PCR should be used for specimens for which there is histopathological evidence of invasive fungal infection
  • •If the PCR is positive, sequencing should be applied for fungal speciation

Therapeutic drug monitoring (TDM)

• •Recommendations for TDM apply as per previous best practice, with possible modifications for isavuconazole TDM in patients in intensive care units and potentially both isavuconazole and posaconazole in those on long-term therapy ⁷⁸
  • •Routine posaconazole TDM is not required in most individuals taking the tablet or intravenous formulations

Histopathology

• •Recommendations for histology testing apply as per previous best practice²
  • •Reporting standards were described in detail in 2015, including telephoning positive results to clinicians
• •Conduct standard and triple specialised stains on all tissue, if fungal disease is suspected²
• •Additional specialised stains, such as mucicarmine, for suspected cryptococcosis and Fontana Masson for dematiaceous moulds should be considered and have been added in the 2025 guidance
• •Request all stains immediately (ie, do not delay until haematoxylin and eosin sections have been viewed) on tissue from immunocompromised patients who are suspected of fungal disease²

Radiology

• •Recommendations for radiology apply as per previous best practice²
• •Patients with leukaemia and allogenic haematopoietic stem cell transplant recipients with any suspicion of invasive fungal infection should have a CT scan of the chest within 48 h
• •Patients who are immunocompromised with any neurological features should have an MRI scan of the brain within 48 h
• •Patients with suspected invasive paranasal sinus fungal infection (including mucormycosis) should have a non-contrast CT scan of the sinuses within 48 h
  • •Consider also imaging sinuses and brain in those treated with ibrutinib

Clinical management

• •Clinical recommendations apply as per previous best practice²
• •All intravascular devices should be removed promptly if clinically feasible after diagnosis of candidaemia

Table. Scope of the 2025 and 2015 recommendations by topic

Empty Cell	Inclusion of guidance		Comment	Location within this Review
Empty Cell	2015	2025	Empty Cell	Empty Cell
Radiology	Yes	No	2015 guidance broadly unchanged	Panel p 6
Histopathology reporting	Yes	No	2015 guidance largely unchanged apart from the additional guidance on mucicarmine and Fontana Masson	Panel p 6
Clinical requests and reporting	Yes	Yes	Updated with a focus on turnaround time and antifungal stewardship	Appendix pp 17, 27; Review pp 10, 11
Cryptococcal antigen	Yes	Yes	2015 guidance undated to include bronchoalveolar lavage testing	Panel p 5; Review p 4
Clinical management	Yes	Yes	Main addition is handling of intravenous catheters	Appendix pp 20–22
Paediatric patients	No	Yes	Assay performance data summarised	Appendix pp 9, 26
Direct microscopy	Yes	Yes	Updated, with emphasis on fluorescent brightener use	Appendix p 1
Fungal culture	Yes	Yes=	Updated with focus on mycology-specific procedures, including handling of different samples and high-volume respiratory cultures	Apendix pp 2–5
Fungus identification	Yes	Yes	Updated: MALDI ToF, reporting nomenclature, and guidance on when to speciate yeasts	Appendix pp 5, 6
Susceptibility testing	Yes	Yes	Updated with additional methodological advice, and use of anidulafungin for all echinocandin testing	Appendix pp 6, 7
1,3-β-d-glucan testing	Yes	Yes	Updated and expanded	Review pp 4, 6, 7; Panel p 5
Aspergillus antigen	Yes	Yes	Updated with more cutoff data and mention of lateral flow devices	Appendix pp 17, 20, 26, 27; Review p 7; Panel p 5
Histoplasma antigen	No	Yes	Brief mention	Review p 7
Candida antigen	No	Yes	Brief mention	Appendix p 28
Pneumocystis PCR	Yes	Yes	Major update	Appendix pp 7, 25, 30; Review p 8; Panel p 6
Aspergillus PCR	No	Yes	Assay performance and utility	Appendix p 25; Review p 8; Panel p 6
Candida PCR	No	Yes	Assay performance and utility	Appendix p 25, Review p 9; Panel p 6
Mucorales PCR	No	Yes	Assay performance and utility	Appendix p 25; Review p 9; Panel p 6
Pan-fungal identification	No	Yes	For unfixed or fixed tissue	Appendix p 25; Review p 9; Panel p 6
Quality of evidence summary	No	Yes	Primarily for molecular diagnostics	Appendix p 25
Global adaptation for high-income to low-income economies	No	Yes	To distinguish essential tests for all laboratories from those recommended for referral centre and specialised laboratories	Appendix pp 30–32
Aspergillus IgG antibody	Yes	Yes	Expanded slightly, much additional data published	Appendix p 30; Panel p 6
Aspergillus IgE antibody	Yes	Yes	Expanded slightly	Appendix p 31; Panel p 6; Review pp 7, 8
Candida antibody	No	Yes	Brief mention	Appendix p 28
Recommendations including children	No	Yes	Blood cultures, non-culture tests, fungal biomarkers	Appendix p 26
Therapeutic drug monitoring	Yes	Yes	Inclusion of isavuconazole; the 2014 British Society of Medical Mycology guidance is in the process of being updated	Appendix p 28
External quality assurance schemes	No	Yes	New addition	Appendix p 29
Turnaround time	Yes	Yes	Updated in detail, including reporting positives to clinicians within 2 h for tests diagnosing invasive fungal infections	Appendix p 27