Fungal Biomarkers and Molecular Diagnostics
Biomarkers and molecular tests detect fungal components (cell wall polysaccharides, capsular antigen, DNA) or the host response (antibody) in blood, cerebrospinal fluid, urine, and respiratory specimens. They are faster and more sensitive than culture and are often the only evidence of invasive disease.
Each test has limitations in sensitivity, specificity, host group, and prior antifungal use, so the pre-test probability, the specimen, and the timing determine the interpretation. A single negative test rarely excludes disease, and combinations and serial testing improve performance.
| Test | Target | Specimen | Principal use |
|---|---|---|---|
| Galactomannan (enzyme immunoassay, lateral flow) | Aspergillus cell wall polysaccharide | Serum, bronchoalveolar lavage, cerebrospinal fluid | Invasive aspergillosis in haematology and some other hosts |
| Beta D glucan | Beta 1,3 D glucan in the cell wall of most fungi | Serum, plasma | Invasive candidiasis, aspergillosis, pneumocystis pneumonia, and others (not cryptococcosis or mucormycosis) |
| Cryptococcal antigen (lateral flow, latex, enzyme immunoassay) | Capsular glucuronoxylomannan | Serum, plasma, cerebrospinal fluid, whole blood | Cryptococcosis, and screening in HIV |
| Histoplasma antigen | Galactomannan | Urine, serum, bronchoalveolar lavage, cerebrospinal fluid | Histoplasmosis |
| Blastomyces and Coccidioides antigens | Galactomannan | Urine, serum, cerebrospinal fluid | Blastomycosis and coccidioidomycosis |
| Talaromyces antigen (Mp1p) | Mannoprotein | Serum, plasma, urine | Talaromycosis |
| Candida mannan and anti mannan | Mannan antigen and antibody | Serum | Invasive candidiasis (adjunct) |
| T2 magnetic resonance | Candida DNA | Whole blood | Candidaemia in 3 to 5 hours |
| Polymerase chain reaction (specific) | Aspergillus, Mucorales, Candida, Pneumocystis, Histoplasma, and others | Blood, bronchoalveolar lavage, tissue, cerebrospinal fluid | Rapid detection and resistance markers |
| Panfungal polymerase chain reaction and sequencing | Internal transcribed spacer, large subunit ribosomal DNA | Tissue, fluids | Identification of unusual fungi |
| Metagenomic sequencing | Total nucleic acid | Cerebrospinal fluid, bronchoalveolar lavage, plasma | Unknown or mixed infection |
| Antibody tests (precipitin, complement fixation, enzyme immunoassay, immunodiffusion) | Host IgG and IgM | Serum, cerebrospinal fluid | Chronic pulmonary aspergillosis, endemic mycoses, and allergic disease |
| Mass spectrometry | Protein profile | Colonies, blood culture pellets | Identification |
| Peptide nucleic acid fluorescence in situ hybridisation | Ribosomal RNA | Positive blood cultures | Rapid species identification of Candida |
| Aspect | Details |
|---|---|
| Principle | A sandwich enzyme immunoassay for galactomannan, a polysaccharide released by growing Aspergillus hyphae (and by some other fungi) |
| Cut off | Optical density index 0.5 or higher in serum (two consecutive positive samples increase specificity; 1.0 to 1.5 is sometimes used for higher specificity), and 1.0 or higher in bronchoalveolar lavage (0.8 in some assays) |
| Performance | Serum in neutropenic haematology patients: sensitivity about 70 to 80 percent and specificity about 85 to 90 percent; bronchoalveolar lavage: sensitivity about 80 to 90 percent, specificity about 90 percent; lower sensitivity in solid organ transplant, in non neutropenic patients, in chronic pulmonary disease, and in patients on mould active prophylaxis (below 30 percent) |
| Use | Twice weekly screening in high risk haematology patients not receiving mould active prophylaxis; diagnostic test in patients with suspected pulmonary or cerebral aspergillosis (cerebrospinal fluid, bronchoalveolar lavage); monitoring of response (a falling level indicates response) |
| False positives | Cross reactive fungi (Fusarium, Talaromyces, Histoplasma, Cryptococcus, Penicillium, Paecilomyces, Alternaria), some intravenous solutions (older piperacillin tazobactam batches, plasma electrolyte solutions with gluconate), amoxicillin clavulanate, intravenous immunoglobulin, and food products (neonatal formula, soy), gut translocation in mucositis, and bifidobacteria colonisation in neonates |
| False negatives | Mould active antifungal drugs, a low burden of angioinvasive disease (in non neutropenic patients), localised disease, and non galactomannan secreting species |
| Lateral flow assay | A point of care test with a monoclonal antibody (IMMY), on serum, bronchoalveolar lavage, and urine; fast (under 1 hour); sensitivity in bronchoalveolar lavage about 80 to 90 percent; performance in serum is similar to the enzyme immunoassay in haematology |
| Not recommended | Screening in patients on mould active prophylaxis, and a single positive serum result without imaging in a low risk patient |
| Aspect | Details |
|---|---|
| Principle | A colourimetric or turbidimetric assay (Fungitell uses the Limulus amoebocyte lysate factor G pathway) for a polysaccharide of the cell wall of most fungi |
| Cut off | 80 picograms per mL or higher (positive), 60 to 79 (indeterminate), and below 60 (negative) with Fungitell; two consecutive positive samples increase specificity |
| Performance | For invasive fungal disease, sensitivity about 75 to 80 percent and specificity about 80 to 85 percent; high negative predictive value (above 90 percent) in haematology patients; sensitivity for pneumocystis pneumonia about 90 to 95 percent, and for invasive candidiasis about 75 to 80 percent |
| Detects | Candida, Aspergillus, Pneumocystis jirovecii, Fusarium, Scedosporium, Histoplasma, Coccidioides (variable), and Talaromyces |
| Does not detect | Mucorales, Cryptococcus (low and variable amounts of glucan in the capsule), Blastomyces (low), and Candida parapsilosis (poorly) |
| False positives | Intravenous immunoglobulin, albumin, and blood products (glucan contaminated), haemodialysis with cellulose membranes, surgical gauze and sponges, glucan containing antibacterials (some beta lactams), bacteraemia (Gram positive and Pseudomonas), mucositis, and sample contamination |
| Use | An adjunct in the diagnosis of invasive candidiasis (negative tests can allow early stopping of empirical antifungal therapy), pneumocystis pneumonia (a negative test makes the diagnosis very unlikely), and invasive aspergillosis (with galactomannan) |
| Limits | Non specific for the fungal species; repeated positive tests with a compatible picture are required |
| Aspect | Details |
|---|---|
| Methods | Lateral flow assay (the preferred test, a point of care and low cost device), latex agglutination, and enzyme immunoassay |
| Performance | In cerebrospinal fluid, sensitivity and specificity above 99 percent for cryptococcal meningitis; in serum in HIV, sensitivity near 100 percent; sensitivity is lower in localised pulmonary disease and low burden disease in non HIV |
| Quantitation | Titres (or semi quantitative lateral flow grading) track fungal burden, with high titres of 1 in 160 or more predicting meningitis and a poor outcome; titres are not used to monitor response, because they fall slowly |
| Prozone effect | Very high antigen concentrations can give false negative results in latex and lateral flow tests, and dilution of the sample solves it |
| False positives | Rare: Trichosporon, Capnocytophaga, and Stomatococcus; contamination of the sample with soap or disinfectants and rheumatoid factor in some latex assays |
| Screening | Serum or plasma or finger stick whole blood in HIV with CD4 below 100 (or 200) cells per microlitre, followed by lumbar puncture for positive tests, and pre-emptive antifungal therapy |
| Cerebrospinal fluid | Antigen, India ink, culture, and opening pressure in all suspected cases |
| Test | Details |
|---|---|
| Histoplasma antigen | Urine sensitivity about 95 percent in progressive disseminated disease and 80 percent in acute pulmonary disease, with cross reaction with Blastomyces, Coccidioides, Paracoccidioides, Talaromyces, and Emergomyces; used for monitoring, and a lateral flow version is available |
| Blastomyces antigen | Urine and serum enzyme immunoassay with sensitivity about 76 to 93 percent, and cross reaction with Histoplasma |
| Coccidioides antigen | Enzyme immunoassay on urine, serum, and cerebrospinal fluid, with sensitivity about 70 percent in disseminated disease |
| Talaromyces Mp1p antigen | Serum or plasma enzyme immunoassay, sensitivity about 86 percent and specificity above 95 percent |
| Candida mannan and anti mannan | Together, sensitivity 80 to 85 percent and specificity 85 percent; used mainly in Europe |
| Pneumocystis tests | Beta D glucan, immunofluorescence, and polymerase chain reaction |
| Sporothrix and others | Antibody tests, with limited availability |
| Test | Use |
|---|---|
| Aspergillus specific IgG (precipitins, enzyme immunoassay) | Chronic pulmonary aspergillosis (sensitivity above 90 percent), allergic bronchopulmonary aspergillosis, and aspergilloma; a threshold of 40 milligrams of antigen specific antibody per litre or higher |
| Aspergillus specific IgE and total IgE, skin prick test | Allergic bronchopulmonary aspergillosis and sensitisation |
| Endemic mycoses (immunodiffusion, complement fixation, enzyme immunoassay) | Coccidioidomycosis, histoplasmosis, blastomycosis, and paracoccidioidomycosis; limits in the immunocompromised and in early disease |
| Candida antibodies | Limited value |
| Cerebrospinal fluid antibody | Coccidioidal meningitis (complement fixing antibody) |
| Test | Details |
|---|---|
| Aspergillus polymerase chain reaction | Blood (serum or plasma) and bronchoalveolar lavage; sensitivity about 80 to 90 percent and specificity about 75 to 95 percent in haematology patients; combined with galactomannan, the sensitivity is above 95 percent; commercial and in house assays, and a standardised method improves reproducibility; some assays detect azole resistance mutations (TR34/L98H, TR46/Y121F/T289A in the cyp51A gene) |
| Mucorales polymerase chain reaction | Blood and bronchoalveolar lavage; sensitivity about 85 to 90 percent in blood and positive 3 to 9 days before imaging in some studies; useful when galactomannan is negative |
| Candida tests | Polymerase chain reaction on blood (sensitivity 90 percent, specificity 90 to 95 percent), T2 magnetic resonance (limit of detection 1 to 3 colony forming units per mL; species groups Candida albicans or Candida tropicalis, Candida parapsilosis, Candida glabrata or Candida krusei, and Candida auris; reported in 3 to 5 hours), and rapid identification from positive blood cultures (peptide nucleic acid fluorescence in situ hybridisation, multiplex polymerase chain reaction panels, and mass spectrometry) |
| Candida auris polymerase chain reaction | Screening swabs (axilla and groin) and culture with selective enrichment for infection control |
| Pneumocystis quantitative polymerase chain reaction | Bronchoalveolar lavage and upper respiratory tract specimens; very high negative predictive value |
| Syndromic panels | Multiplex panels for meningoencephalitis (including Cryptococcus; lower sensitivity than the antigen test), respiratory pathogens, and blood culture identification |
| Panfungal polymerase chain reaction and sequencing | Targets the internal transcribed spacer region and the large subunit ribosomal DNA of tissue, fluids, and formalin fixed samples; identifies unusual fungi; at risk of contamination, so interpretation requires a compatible picture |
| Metagenomic next generation sequencing | Cerebrospinal fluid, bronchoalveolar lavage, and plasma cell free DNA; identifies unexpected and mixed pathogens; costly, and needs experts to separate colonisation and contamination |
| Antifungal resistance markers | cyp51A (azole resistant Aspergillus fumigatus), FKS1 and FKS2 hot spot mutations (echinocandin resistant Candida), ERG11 mutations (fluconazole resistant Candida), and TAC1B and others in Candida auris |
| Whole genome sequencing | Outbreak investigation and clade identification for Candida auris, with tracing of transmission |
| Mass spectrometry for resistance | Under investigation |
| Limits | Cost, availability, contamination, the difficulty of separating colonisation from infection, and the need to confirm with clinical and imaging data; lack of standardisation for many assays |
| Clinical scenario | Approach |
|---|---|
| Neutropenic haematology patient not on mould active prophylaxis | Serum galactomannan twice weekly (with or without Aspergillus polymerase chain reaction) combined with chest computed tomography when fever persists; bronchoalveolar lavage with galactomannan, culture, and polymerase chain reaction for new infiltrates |
| Patient on mould active prophylaxis | Galactomannan is poorly sensitive, so use computed tomography, bronchoalveolar lavage (galactomannan, polymerase chain reaction, culture), and clinical suspicion |
| Suspected invasive candidiasis in intensive care | Blood cultures (repeated), beta D glucan (a negative test makes disease unlikely and supports stopping empirical therapy), T2 magnetic resonance or polymerase chain reaction where available |
| Suspected pneumocystis pneumonia | Beta D glucan (above 80 picograms per mL) with bronchoalveolar lavage polymerase chain reaction or immunofluorescence; a negative result in both makes the diagnosis unlikely |
| Suspected mucormycosis | Tissue (microscopy, histology, culture), Mucorales polymerase chain reaction on blood and tissue, and negative galactomannan and beta D glucan to separate from aspergillosis |
| HIV with headache or advanced disease | Cryptococcal antigen on serum and cerebrospinal fluid; Histoplasma, Talaromyces, and Emergomyces tests by region |
| Endemic mycosis suspected | Antigen in urine, serum, and bronchoalveolar lavage, serology, culture, and histology |
| Chronic cavitary lung disease | Aspergillus specific IgG, culture, and galactomannan in sputum or lavage |
| Unusual or refractory disease | Tissue for culture, panfungal polymerase chain reaction, and sequencing |
Combining tests
| Combination | Effect |
|---|---|
| Galactomannan with beta D glucan | Two positive results increase specificity (above 95 percent); a concordant negative result has a high negative predictive value |
| Serum galactomannan with polymerase chain reaction | Increases sensitivity (above 95 percent) and shortens time to diagnosis |
| Antigen with imaging and culture | A multi test approach is used in practice, because no single test is adequate |
| Serial testing | A falling biomarker (galactomannan, beta D glucan, antigen) tracks response, and a rising result predicts failure or relapse |
Laboratories validate each assay against their patient population, report a numerical value along with the cut off, indicate false positive causes, and offer advice on interpretation. Tests are ordered in a defined clinical setting rather than for screening in low risk patients, because a low pre-test probability gives poor positive predictive value.
A negative biomarker does not exclude disease in hosts with a low burden, in localised disease, or on active therapy. Treatment decisions combine the host factors, clinical and imaging findings, mycological evidence, and the biomarkers, in line with the established definitions of proven, probable, and possible invasive fungal disease (host factor, clinical features, and mycological evidence).
