Research Update: Panfungal PCR improves tissue diagnosis—but targeted testing remains essential

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Diagnosing invasive fungal disease from tissue remains difficult. Culture can be slow and insensitive, while histopathology may show fungal elements without identifying the organism. Two newly published studies underline both the value of molecular testing and the importance of using it selectively.

Panfungal PCR performs well on tissue—but laboratories need common standards

A systematic review and meta-analysis from the Fungal PCR Initiative assessed panfungal PCR amplicon sequencing on tissue specimens. The analysis included 28 studies and examined both formalin-fixed paraffin-embedded (FFPE) tissue and fresh or frozen samples.

For FFPE tissue, pooled sensitivity was 75.4% and specificity was 93.5%. Sensitivity was higher in non-fixed tissue, at 86.5%. In the studies directly comparing methods, panfungal PCR was substantially more sensitive than culture: 88.2% versus 52.2%.

These figures support the use of panfungal PCR to help identify fungi in tissue, particularly where histopathology has demonstrated fungal elements but culture is negative or unavailable. It is not a replacement for conventional testing: the authors recommend its use alongside histopathology, culture and, where appropriate, fungus-specific PCR assays.

The important limitation is variation. Laboratories use different extraction methods, PCR targets and analytical approaches, and the review could not identify one target as clearly superior. Further inter-laboratory work is needed to standardise methods and improve reproducibility.

More sequencing is not automatically better sequencing

A separate real-world study of internal transcribed spacer (ITS) sequencing provides a useful counterbalance. Researchers assessed 315 direct clinical specimens from 227 patients in a university hospital where clinicians could order the test without restriction.

ITS sequencing had high specificity (96.9%), but sensitivity was only 35.6%. Positive results took a median of 12 days, and the test changed antifungal management in only two patients—0.9% of the study population. Many culture-negative but ITS-positive bronchoalveolar lavage samples appeared to represent colonisation rather than invasive infection.

The finding is not that ITS sequencing has no role. Rather, it highlights the need for diagnostic stewardship: selecting the right patient, specimen and clinical question before ordering a broad molecular test. Testing used indiscriminately can produce results too late, or findings that are difficult to interpret, without improving immediate care.

What this means in practice

Molecular identification from tissue is becoming an increasingly valuable part of the invasive fungal disease diagnostic pathway. The strongest current evidence supports panfungal PCR as an adjunct when tissue is available, especially when histopathology suggests fungal infection and culture has failed to provide an answer.

However, molecular results must always be interpreted with the clinical picture, radiology, histopathology and other mycological evidence. A technically positive result is not, by itself, proof of invasive disease.

References

  1. Filippidis P, El Khoury C, Cruciani M, et al. Diagnostic performance of panfungal PCR on tissue specimens for the diagnosis of invasive fungal diseases: a systematic review and meta-analysis of the Fungal PCR Initiative (FPCRI). Journal of Clinical Microbiology. 2026. doi: 10.1128/jcm.00721-26.
  2. Techopitch V, Santanirand P, Phuphuakrat A. Diagnostic yield and impact of internal transcribed spacer sequencing from clinical specimens on antifungal management in patients with suspected fungal infection. Microbiology Spectrum. 2026. doi: 10.1128/spectrum.03964-25.