Fungal Diagnostics Weekly Update: 31 March – 7 April 2026

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This week’s fungal diagnostics literature highlights continued progress in molecular testing, growing interest in biomarker development, and ongoing real-world problems such as over-testing, unequal access to diagnostics, and confusion between aspergillosis and other lung diseases. Overall, the picture remains familiar: newer methods are improving detection, but no single test is sufficient on its own.

Key Highlights

  • PCR continues to strengthen as a diagnostic tool, including new multi-species Aspergillus assays.
  • Droplet digital PCR (ddPCR) may improve sensitivity in difficult infections.
  • Eosinophil-derived neurotoxin (EDN) is emerging as a possible biomarker for allergic bronchopulmonary aspergillosis (ABPA).
  • Metagenomic sequencing is expanding, but still faces interpretation and false-positive challenges.
  • Real-world diagnostic problems remain important, including TB overlap, galactomannan over-testing, and variation in test availability.
  • Specialist and advanced methods continue to show value where conventional tests are negative or unclear.

Contents

1. Molecular Diagnostics

Multi-species Aspergillus PCR validated

A clinical validation study reported a new reverse transcriptase quantitative PCR (RT-qPCR) assay capable of detecting multiple Aspergillus species rather than focusing narrowly on Aspergillus fumigatus alone.

Why this matters: This supports the continued maturation of PCR as an important fungal diagnostic tool. Broader species detection may improve performance in invasive aspergillosis and chronic pulmonary aspergillosis (CPA), particularly when non-fumigatus species are involved.

Paper: Benazra M, Mallek W, Ghelfenstein-Ferreira T, Sturny-Leclère A, Dellière S, Hamane S, Lortholary O, Lanternier F, Alanio A; OPTIFIL working group. Clinical validation of a new multi-Aspergillus species reverse transcriptase quantitative PCR: a diagnostic case-control study. Clin Microbiol Infect. 2026. PubMed

Droplet digital PCR improves sensitivity

A duplex droplet digital PCR (ddPCR) assay was reported to improve diagnosis of Aspergillus and Fusarium keratitis compared with standard qPCR approaches.

Why this matters: ddPCR may be particularly useful where fungal burden is low or where conventional PCR struggles with sensitivity. Although this study was in ocular infection, the broader diagnostic principle is important and may eventually influence respiratory fungal diagnostics as well.

Paper: Tawde Y, Das S, Singh S, Basak S, Sharma S, Gupta A, Bal A, Rudramurthy SM, Yadav G, Ghosh A. Duplex droplet digital PCR (ddPCR) improves the diagnosis of Aspergillus and Fusarium keratitis. Exp Eye Res. 2026. PubMed

Next-generation sequencing reveals greater fungal complexity in cystic fibrosis airways

A study of cystic fibrosis airway specimens applied next-generation sequencing (NGS) to detect and quantify fungal communities that are often missed by culture-based methods.

Why this matters: Standard culture may underestimate the range and abundance of fungi present in airway samples. Molecular profiling could improve understanding of mixed colonisation, microbial interactions, and the wider airway mycobiome.

Paper: Fought MK, Hong G, Quinn AC, Wagner BD, Robertson CE, Harris JK, Laguna TA. Molecular Methods to Detect and Quantify Fungal Communities in Cystic Fibrosis Airway Specimens. Pediatr Pulmonol. 2026. PubMed

Metagenomic sequencing continues to expand, but interpretation remains difficult

Several papers this week reinforced the growing role of metagenomic next-generation sequencing (mNGS) in difficult fungal diagnostic scenarios. One report described rapid diagnosis of a mixed pulmonary infection including Aspergillus fumigatus using BAL sequencing, while another assessed real-time mNGS analysis in respiratory samples and highlighted false positives and interpretation challenges.

Why this matters: mNGS is increasingly valuable in complex or culture-negative infections, particularly in immunocompromised patients. However, it still requires careful clinical interpretation and is not yet a simple stand-alone replacement for established diagnostic pathways.

Papers:

  • Wang L, Wang J, Zhu Q, Zhang Q, Qian J. Rapid diagnosis of a mixed pulmonary infection with Rhizopus microsporus, Aspergillus fumigatus, Pneumocystis jirovecii, and Cytomegalovirus in a Lymphoma patient using metagenomic next-generation sequencing: A case report. Diagn Microbiol Infect Dis. 2026. PubMed
  • Boutin S, Klein S, Untergasser G, Loka TP, Jakob S, Caf Y, Khatamzas E, Knabl L, Wrettos G, Knobloch H, Nurjadi D. Evaluating Seqstant LiveGene Analysis in real-time assessment of metagenomic next-generation sequencing (mNGS) data from respiratory samples. Infection. 2026. PubMed | Free PMC article

2. Biomarkers

Eosinophil-derived neurotoxin (EDN) as a possible biomarker for ABPA

A study in Journal of Asthma and Allergy examined eosinophil-derived neurotoxin (EDN) as a potential biomarker for allergic bronchopulmonary aspergillosis (ABPA).

Why this matters: ABPA diagnosis still relies on combining clinical history, immunology, and imaging findings. A useful biomarker could improve diagnostic confidence and potentially help with monitoring disease activity. EDN appears promising, though further validation will still be needed before routine use.

Paper: Zou M, Pan Y, Yang Y, Liu X, Zeng R, Dong L. Eosinophil-Derived Neurotoxin: A Potential Biomarker for Allergic Bronchopulmonary Aspergillosis. J Asthma Allergy. 2026. PubMed | Free PMC article

Biomarker development remains an important unmet need

A broader review discussed the challenge of developing prognostic biomarkers for invasive fungal infections, highlighting the current lack of robust tools for patient stratification and treatment guidance.

Why this matters: While diagnostic biomarkers are improving, there remains a major gap in biomarkers that help predict severity, guide escalation, or identify which patients may do poorly. This remains an important priority across fungal disease.

Paper: Elhaj Mahmoud D, Briard B, Papon N. Developing prognostic biomarkers for invasive fungal infections. Trends Mol Med. 2026. PubMed

3. Real-World Diagnostic Challenges

TB and Aspergillus overlap remains a practical diagnostic problem

A study of patients clinically suspected of pulmonary tuberculosis found respiratory tract Aspergillus infection in the same setting, using microscopy and culture methods.

Why this matters: Pulmonary aspergillosis and TB often share symptoms and radiological features, and this overlap continues to complicate diagnosis. This is particularly relevant for chronic pulmonary aspergillosis, which can be missed or delayed if fungal disease is not actively considered.

Paper: Rahat T, Afroz S, Naheen CR, Sani UIJ, Haider SB, Nazrul AK. Profile of Respiratory Tract Aspergillus Infection among Clinically Suspected Pulmonary Tuberculosis Cases in a Tertiary Care Hospital. Mymensingh Med J. 2026. PubMed

Galactomannan over-testing may reduce value and increase cost

A retrospective study from a tertiary care hospital examined over-testing of galactomannan in haematology patients and found a very low diagnostic return.

Why this matters: This raises an important real-world issue: diagnostics are most useful when well targeted. Blanket or poorly selected testing can produce low yield, unnecessary cost, and confusion. The study supports more selective and clinically guided use of galactomannan.

Paper: Bozkurt I. Over-testing Galactomannan in patients with hematological malignancies: A retrospective analysis from a tertiary care university hospital in Turkiye. Diagn Microbiol Infect Dis. 2026. PubMed

Access to fungal diagnostics remains uneven

A survey of Australasian clinical mycology laboratories showed wide access to some key tests such as galactomannan and PCR, but much lower on-site capacity for mould diagnostics and susceptibility testing.

Why this matters: Even where important tests technically exist, access may depend on referral pathways, outsourcing, or specialist centre support. This reinforces the value of expert laboratory networks and referral models.

Paper: Stewart AG, Douglas AP, Kidd SE, Salmanton-García J, Cornely OA, Morris AJ, Halliday CL, Chen SC; Australia and New Zealand Mycoses Interest Group. Mycological testing in Australasian clinical mycology laboratories: survey of the current state and access to testing. Pathology. 2026. PubMed

4. Emerging and Specialist Diagnostics

mNGS as a rescue tool when standard tests are negative

A case of postoperative ocular fungal infection demonstrated that standard cultures and PCR were negative, but metagenomic RNA deep sequencing identified Rhizopus stolonifer.

Why this matters: This is a strong example of sequencing acting as a rescue diagnostic tool where routine methods fail. Although not an aspergillosis case, the principle is directly relevant to invasive mould disease more broadly.

Paper: Turner ML, Nguyen MT, Kung Y, Doan T, Seitzman GD. Rhizopus angle abscess, scleritis and endophthalmitis following Kahook Dual Blade goniotomy and phacoemulsification. Am J Ophthalmol Case Rep. 2026. PubMed | Free PMC article

Advanced imaging may help identify rare fungal infections

A corneal case report highlighted how in vivo confocal microscopy and histopathology can facilitate diagnosis of rare fungal infection.

Why this matters: Specialist imaging techniques may have a valuable role in selected settings, especially ophthalmology, where rapid identification can be sight-saving.

Paper: Chen AC, Ghaffari R, Glasgow BJ. Recognition of Bulbilli Facilitates the Diagnosis of Cladorrhinum bulbillosum. Cornea. 2026. PubMed

5. Case-Based Diagnostic Insights

Invasive aspergillosis can still present outside classic risk groups

A case report described invasive pulmonary aspergillosis in an immunocompetent patient following occupational exposure to iron oxide dust. Diagnosis involved bronchoscopy and microbiological testing.

Why this matters: Clinical suspicion remains important. Although invasive aspergillosis is strongly associated with immunosuppression, unusual exposures and local lung damage may still create opportunities for infection in other patients.

Paper: Zhou C, Chao L, Guo C, Zhou Y. Invasive Pulmonary Aspergillosis in an Immunocompetent Patient Following Occupational Exposure to Iron Oxide Dust: A Long-Term Follow-Up Case Report. Infect Drug Resist. 2026. PubMed | Free PMC article

Paediatric invasive tracheobronchial aspergillosis diagnosed by BAL sequencing

A paediatric case series described invasive necrotizing tracheobronchial aspergillosis in children, with next-generation sequencing of bronchoalveolar lavage fluid detecting Aspergillus flavus in one case.

Why this matters: This supports the value of BAL-based molecular diagnostics in difficult paediatric cases and shows how sequencing can contribute where diagnosis is otherwise delayed or uncertain.

Paper: Jin F, Tao X, Wu H, Wu L, Wang Y. Invasive Necrotizing Tracheobronchial Aspergillosis in Children: A Case Series and Literature Review. Am J Case Rep. 2026. PubMed | Free PMC article

6. Overall Interpretation

No single test is enough

This week’s literature again supports a multi-modal approach. Culture, microscopy, galactomannan, PCR, sequencing, and imaging all have roles, but none is sufficient in every case.

Molecular diagnostics are advancing quickly

PCR is becoming more standardised, ddPCR may improve sensitivity further, and sequencing continues to expand into complex cases. These methods are increasingly important, but still require careful interpretation.

Biomarkers remain promising but incomplete

EDN is a good example of progress in ABPA, but the broader biomarker field still lacks a robust tool that can reliably guide prognosis or treatment across fungal disease.

Service organisation still matters

Several studies this week highlighted the importance of targeted testing, specialist expertise, and access to advanced diagnostics. This has direct relevance for referral pathways and specialist fungal services.

References

  1. Zou M, Pan Y, Yang Y, Liu X, Zeng R, Dong L. Eosinophil-Derived Neurotoxin: A Potential Biomarker for Allergic Bronchopulmonary Aspergillosis. J Asthma Allergy. 2026. PubMed | Free PMC article
  2. Benazra M, Mallek W, Ghelfenstein-Ferreira T, Sturny-Leclère A, Dellière S, Hamane S, Lortholary O, Lanternier F, Alanio A; OPTIFIL working group. Clinical validation of a new multi-Aspergillus species reverse transcriptase quantitative PCR: a diagnostic case-control study. Clin Microbiol Infect. 2026. PubMed
  3. Tawde Y, Das S, Singh S, Basak S, Sharma S, Gupta A, Bal A, Rudramurthy SM, Yadav G, Ghosh A. Duplex droplet digital PCR (ddPCR) improves the diagnosis of Aspergillus and Fusarium keratitis. Exp Eye Res. 2026. PubMed
  4. Elhaj Mahmoud D, Briard B, Papon N. Developing prognostic biomarkers for invasive fungal infections. Trends Mol Med. 2026. PubMed
  5. Fought MK, Hong G, Quinn AC, Wagner BD, Robertson CE, Harris JK, Laguna TA. Molecular Methods to Detect and Quantify Fungal Communities in Cystic Fibrosis Airway Specimens. Pediatr Pulmonol. 2026. PubMed
  6. Rahat T, Afroz S, Naheen CR, Sani UIJ, Haider SB, Nazrul AK. Profile of Respiratory Tract Aspergillus Infection among Clinically Suspected Pulmonary Tuberculosis Cases in a Tertiary Care Hospital. Mymensingh Med J. 2026. PubMed
  7. Turner ML, Nguyen MT, Kung Y, Doan T, Seitzman GD. Rhizopus angle abscess, scleritis and endophthalmitis following Kahook Dual Blade goniotomy and phacoemulsification. Am J Ophthalmol Case Rep. 2026. PubMed
  8. Zhou C, Chao L, Guo C, Zhou Y. Invasive Pulmonary Aspergillosis in an Immunocompetent Patient Following Occupational Exposure to Iron Oxide Dust: A Long-Term Follow-Up Case Report. Infect Drug Resist. 2026. PubMed
  9. Jin F, Tao X, Wu H, Wu L, Wang Y. Invasive Necrotizing Tracheobronchial Aspergillosis in Children: A Case Series and Literature Review. Am J Case Rep. 2026. PubMed
  10. Stewart AG, Douglas AP, Kidd SE, Salmanton-García J, Cornely OA, Morris AJ, Halliday CL, Chen SC; Australia and New Zealand Mycoses Interest Group. Mycological testing in Australasian clinical mycology laboratories: survey of the current state and access to testing. Pathology. 2026. PubMed
  11. Bozkurt I. Over-testing Galactomannan in patients with hematological malignancies: A retrospective analysis from a tertiary care university hospital in Turkiye. Diagn Microbiol Infect Dis. 2026. PubMed
  12. Wang L, Wang J, Zhu Q, Zhang Q, Qian J. Rapid diagnosis of a mixed pulmonary infection with Rhizopus microsporus, Aspergillus fumigatus, Pneumocystis jirovecii, and Cytomegalovirus in a Lymphoma patient using metagenomic next-generation sequencing: A case report. Diagn Microbiol Infect Dis. 2026. PubMed
  13. Boutin S, Klein S, Untergasser G, Loka TP, Jakob S, Caf Y, Khatamzas E, Knabl L, Wrettos G, Knobloch H, Nurjadi D. Evaluating Seqstant LiveGene Analysis in real-time assessment of metagenomic next-generation sequencing (mNGS) data from respiratory samples. Infection. 2026. PubMed
  14. Chen AC, Ghaffari R, Glasgow BJ. Recognition of Bulbilli Facilitates the Diagnosis of Cladorrhinum bulbillosum. Cornea. 2026. PubMed