What are the methods for testing fungal infections?

Diagnostic Testing Methods for Fungal Infections

For diagnosing fungal infections, multiple complementary diagnostic tests should be used simultaneously rather than relying on a single test, as no single test has sufficient sensitivity to rule out fungal infection. 1, 2

Direct Visualization and Culture Methods

• Direct microscopy with calcofluor white stain provides rapid visualization of fungal elements and can provide information on important features like the presence of septa, hyphal diameter, or ramification patterns 2
• Tissue samples from patients with suspected invasive fungal disease (IFD) should be examined by both mycological culture and microscopy 1
• Fungal cultures remain the gold standard for identification but have limited sensitivity (30-60%) and may take up to 5 weeks for completion, particularly for molds 2, 1
• Histopathology with special stains (e.g., GMS, PAS, mucicarmine) can help identify fungal elements in tissue samples 1

Antigen Detection Tests

• Galactomannan (GM) testing in serum is strongly recommended for patients with severe immunocompromise suspected of having invasive pulmonary aspergillosis (sensitivity 80-100%, specificity 90-100%) 1, 2
• Bronchoalveolar lavage (BAL) testing with GM is strongly recommended in patients with suspected invasive fungal diseases, especially when serum GM is negative but risk factors are present 1
• (1→3)-β-D-glucan (BDG) assays can detect various fungal infections but should not be relied upon solely for diagnostic decision-making in critically ill patients with suspected invasive candidiasis 1
• Cryptococcal antigen detection in blood and/or CSF is highly indicative (>95%) of cryptococcal infection and should always include parallel testing of CSF and serum 1
• Candida mannan antigen testing (Platelia Candida) may be useful but has variable sensitivity (30-70%) and specificity (88%) 1

Antibody Detection Tests

• Serum antibody testing is recommended for endemic mycoses (blastomycosis, coccidioidomycosis, histoplasmosis) but should be used in conjunction with other diagnostic methods 1
• For blastomycosis, serum antibody testing specifically directed against the anti-BAD-1 antigen is recommended alongside clinical and epidemiological data 1
• Antibody tests for Aspergillus are frequently undetectable in immunocompromised patients and have limited utility 1
• For coccidioidomycosis, EIA panels offer the advantage of local availability with rapid turnaround and higher sensitivity compared to immunodiffusion (ID) and complement fixation (CF) methods, though they are less specific 1

Molecular Diagnostic Methods

• PCR-based assays for Aspergillus are recommended in severely immunocompromised patients suspected of having invasive pulmonary aspergillosis 1, 2
• Pan-fungal PCR assays target conserved fungal DNA regions and can detect multiple fungal species when the causative pathogen is unknown 2
• Molecular testing can be performed on various clinical specimens including blood, serum, and BAL samples with sensitivity ranging from 65-100% depending on specimen type and methodology 2
• PCR testing of BAL samples typically achieves higher sensitivity and specificity than testing blood samples 2

Pathogen-Specific Testing Recommendations

For Aspergillosis:

• Serum and BAL galactomannan testing (sensitivity 74%, specificity 85% at cutoff of 0.5) 2
• Blood or serum Aspergillus PCR testing in severely immunocompromised patients 2
• GM testing should be carried out at least twice a week as circulation of GM in serum is transient 1

For Endemic Mycoses (Blastomycosis, Coccidioidomycosis, Histoplasmosis):

• Multiple diagnostic tests should be used, including direct visualization, culture, urine antigen testing, and serum antibody testing 1
• For blastomycosis, urinary antigen testing is particularly useful in immunocompromised patients but has cross-reactivity with other mycoses 1
• For coccidioidomycosis, both urinary and serum antigen testing are recommended, especially in immunocompromised patients 1

Important Considerations and Pitfalls

• False-positive galactomannan results may occur in patients undergoing chemotherapy or with mucositis due to cross-reactive epitopes from other fungi or bacteria 1, 2
• Antigen tests often show cross-reactivity between different endemic mycoses (e.g., blastomycosis antigen test cross-reacts with histoplasmosis) 1
• Serological testing for coccidioidomycosis can wane over time, and results might become negative after successful treatment 1
• In immunocompromised patients, the sensitivity of serology is lower (84%) compared to immunocompetent patients (95%) 1
• Empiric antifungal therapy may be withheld until further signs of invasive aspergillosis emerge if galactomannan screening is performed regularly 1

Diagnostic Approach Algorithm

1. Begin with high-resolution CT imaging of the lungs within 24 hours of clinical suspicion 2
2. Perform bronchoscopy with BAL targeting areas with radiographic abnormalities 2
3. Send BAL samples for:
   • Direct microscopy with calcofluor white stain 2
   • Fungal culture on appropriate media 2
   • Pathogen-specific antigen testing (e.g., galactomannan for aspergillosis) 1
   • PCR testing when available 2
4. Simultaneously obtain serum for:
   • Antigen testing (galactomannan, β-D-glucan) 1
   • Antibody testing for endemic mycoses 1
   • PCR testing when available 2
5. Consider urine antigen testing for endemic mycoses 1
6. If initial tests are negative but clinical suspicion remains high, repeat testing and consider tissue biopsy 1