This week’s fungal diagnostics research includes two studies of blood-based metagenomic sequencing, an evaluation of tests for catheter-related candidemia, a prospective study of serum Aspergillus PCR and an exploratory Candida biomarker study.

The strongest emerging theme is the potential for non-invasive sequencing tests to provide additional evidence when conventional diagnosis requires bronchoscopy, biopsy or other invasive sampling. However, none of the new tests can yet replace established microbiology, imaging and clinical assessment.

Plasma sequencing detects invasive fungal infections in high-risk children

A retrospective study evaluated plasma cell-free metagenomic next-generation sequencing (mNGS) in children and young adults being investigated for invasive fungal infection.

The researchers reviewed 227 suspected infection episodes involving 180 high-risk patients. Forty-five episodes met EORTC-MSG criteria for proven or probable invasive fungal infection. Plasma mNGS was performed in 36 of these episodes and identified the causative organism in 28, giving a positive percentage agreement of 77.8%. Negative percentage agreement was 90.4%.

Candida and Aspergillus were the fungi most frequently detected. The authors found that plasma mNGS compared favourably with culture and targeted PCR performed on invasively collected samples.

This could be particularly useful when obtaining tissue or respiratory samples is difficult or risky. However, the study was retrospective and conducted at one centre, and mNGS was ordered selectively rather than systematically. Positive and negative percentage agreement should also not automatically be interpreted as sensitivity and specificity.

Clinical significance: Plasma mNGS may provide useful non-invasive evidence of invasive fungal infection, but it should currently supplement rather than replace conventional investigations.

Read the plasma mNGS study in Microbiology Spectrum

Blood mNGS may help distinguish PJP from colonisation

A second study investigated whether blood mNGS could distinguish active Pneumocystis jirovecii pneumonia (PJP) from respiratory colonisation.

The study included 73 patients with suspected PJP who had paired bronchoalveolar lavage fluid and blood mNGS testing. Fifty were classified as having PJP and 23 as having P. jirovecii colonisation.

Using a threshold of more than 4.8 reads per million, blood mNGS produced:

• Sensitivity of 62.0%
• Specificity of 95.7%
• Area under the ROC curve of 0.80

Blood mNGS had a higher reported AUC than BALF mNGS and blood or BALF PCR in this cohort. The amount of P. jirovecii detected in blood was also associated with LDH, C-reactive protein and β-D-glucan levels and was higher among patients who did not survive.

The high specificity is potentially useful: a clearly positive blood result may help support active PJP rather than colonisation. Its sensitivity was much lower, however, meaning that a negative result would miss more than one-third of cases.

BALF also detected a much broader microbial community than blood—216 species compared with 43—so blood testing does not provide equivalent microbiological information.

Clinical significance: Blood mNGS could become a useful non-invasive complementary test for PJP, but its limited sensitivity means that it cannot safely exclude infection.

Read the blood mNGS study in BMC Microbiology

Current tests cannot reliably identify catheter-related candidemia

A prospective two-centre study examined whether paired catheter and peripheral-blood tests could determine whether a central venous catheter was the source of candidemia before the catheter was removed.

The investigators compared β-D-glucan, Candida albicans germ-tube antibodies, mannan antigen, antimannan antibody, T2Candida PCR and the differential time to positivity of blood cultures.

Although 179 candidemia episodes were reviewed, only 28 met the study’s inclusion criteria. Using the stricter definition of catheter-related candidemia, any difference in β-D-glucan between catheter and peripheral blood performed best:

• Sensitivity of 81.82%
• Specificity of 82.35%
• Positive predictive value of 75.00%
• Negative predictive value of 87.50%
• Overall accuracy of 82.14%

Performance declined when stricter β-D-glucan thresholds or a different definition of catheter-related infection were applied. T2Candida and the other biomarkers had sensitivities of only 0–40%, while differential blood-culture time to positivity had limited specificity.

Clinical significance: A catheter–peripheral β-D-glucan difference is an interesting research signal, but no method was sufficiently reliable to replace catheter removal and culture.

Read the catheter-related candidemia study in Open Forum Infectious Diseases

Serum Aspergillus PCR adds sensitivity but has limited specificity

A prospective study assessed an in-house qualitative real-time PCR assay using single serum samples from 87 haematology patients at risk of invasive aspergillosis.

Using the 2020 EORTC/MSGERC definitions, two patients had proven invasive aspergillosis, 37 had probable disease and 48 had possible disease.

When PCR was compared with proven or probable invasive aspergillosis, it produced:

• Sensitivity of 69.23%
• Specificity of 52.08%
• Positive predictive value of 54.00%
• Negative predictive value of 67.57%

Considering either PCR or galactomannan positivity increased sensitivity and negative predictive value to 100%, but specificity remained only 56%.

These findings support using multiple sources of diagnostic evidence, but the combined result requires caution. Only two patients had proven disease, while galactomannan can itself contribute to an EORTC/MSGERC probable-aspergillosis classification. This creates a risk of incorporation bias when galactomannan-containing combinations are evaluated against that classification.

The possible-aspergillosis group also cannot be assumed to represent patients without infection. Consequently, the reported specificity is not equivalent to specificity measured against a clearly disease-negative control group.

Clinical significance: Serum Aspergillus PCR may detect additional cases when combined with galactomannan, but this in-house assay had only moderate sensitivity and low apparent specificity. Results must be interpreted alongside imaging, host factors and other mycological evidence.

Read the Aspergillus PCR study in the Indian Journal of Medical Microbiology

Experimental PRA1 biomarker for Candida vaginitis

An exploratory study examined whether vaginal PRA1—a molecule associated with Candida albicans activity and inflammation—could help distinguish symptomatic vaginitis from clinically insignificant Candida detection.

PRA1 levels were substantially higher in culture-positive and symptomatic samples and correlated with symptom severity. The reported area under the ROC curve was approximately 0.95.

However, the study involved only nine women sampled repeatedly over 12 weeks. Repeated measurements increase the number of observations but do not provide the same evidence as a large cohort of independent patients. The diagnostic thresholds were also developed and evaluated in this very small dataset.

Clinical significance: PRA1 is an interesting candidate marker of biologically active Candida infection, but the current findings are hypothesis-generating and require validation in much larger independent cohorts.

Read the PRA1 study in BMC Microbiology

Overall assessment

The two mNGS studies provide the most important findings this week. Both suggest that microbial cell-free DNA in blood or plasma can contribute useful non-invasive diagnostic evidence. Their performance is not yet sufficient to replace respiratory sampling, tissue diagnosis or established combinations of microbiology, biomarkers, imaging and clinical assessment.

The candidemia study is also clinically valuable because it demonstrates the continuing limitations of available tests for identifying a catheter as the source of infection. The Aspergillus PCR study supports combining diagnostic methods but does not establish the in-house assay as a reliable standalone screening test.

The PRA1 study illustrates a potentially important future direction: detecting fungal activity rather than simply detecting the presence of an organism. Its very small sample means that clinical application remains some distance away.

Note: The blood mNGS and PRA1 reports were released as early, unedited versions of their manuscripts. Details may change during final editorial processing.