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Evidence for Fungal Diagnostics

Learn from this evidence the use of various fungal diagnostic options.

Fungal diagnostic testing ranges from conventional mycological approaches (culture and microscopy) through to the detection of antigens/antibodies and nucleic acids using serological and molecular biomarker assays, respectively. Obviously, in the immunocompromised patient the detection of antibodies will likely be limited by the immune status. In the critically-ill, awaiting the response of humoral immunity in producing antibodies can result in a delay in positivity that restricts the clinical utility of of this approach to patients with chronic/allergic fungal disease.

While culture and microscopy can be applied to a broad range of specimens and detect a wide range of fungi, a reliance on conventional mycological approaches limits testing to confirming infection, the established microbiological strategy. Typically, the sensitivity of culture and microscopy is insufficient (50%), a negative result should not be used to exclude infection, whether this is blood or respiratory culture for the investigation of sepsis and pneumonia, respectively.

Image stating 'only 50% sensitive'

Given the relatively low incidence of invasive fungal disease (IFD <10% in most cohorts), the high pre-test probability of not having infection (>90%) should be exploited by using negative results from highly sensitive test to exclude IFD. The availability of biomarker testing provides sufficient performance to exclude IFD Table 1 but performance can vary depending on sample type, testing strategy, patient population, cause of IFD and the use of antifungal therapy prior to sampling, which has affected both sensitivity and specificity of biomarker testing.

The presence of multiple positive results, whether within a single test type or across multiple different tests, increases the likelihood of IFD. Most tests have been extensively validated in haematology patients compared to critical-care patients, although validation of diagnostics for Pneumocystis pneumonia (PCP) and invasive candidiasis (IC) in the critical-care patient is substantial.
The performance of the Galactomannan ELISA (GM) to aid in the diagnosis of invasive aspergillosis (IA) is good when testing both bronchoalveolar lavage (BAL) fluid and serum Table 1. Galactomannan positivity generates an index value that is proportional to the fungal burden and can be used to monitor AFT response. Sensitivity is compromised by the prior use of AFT, particularly so when testing serum, but positivity in patients receiving AFT is specific for IA. Various sources of false positivity have been documented, including the use of penicillin-based antibiotics, foods and cross-reactivity with other fungi. The performance of serum GM outside the haematology cohort is associated with poor sensitivity, although BAL GM appears comparable.
Aspergillus PCR performance when testing blood and BAL fluid provides slightly superior sensitivity and specificity, respectively, compared to GM Table 1. PCR tests are available to identify genetic markers of resistance direct from the specimen. Concerns over methodological standardisation have been overcome through the efforts of the FPCRI and availability of commercial tests, which make Aspergillus PCR comparable to other biomarker tests. The use of prior AFT has been shown to influence the specificity of Aspergillus PCR.
Candida PCR performance as determined by meta-analysis appears excellent for the diagnosis of candidaemia Table 1. Performance for the detection of other forms of IC require further validation, but appears promising. Candida PCR tests are capable of detecting and differentiating species (C. glabrata, C. krusei and C. auris) associated with AFT resistance direct from the specimen.
Molecular diagnosis of PCP provides excellent sensitivity, sufficient to exclude PCP when negative, particularly when testing deeper respiratory samples (Table 1). Concerns remain over the specificity of the PCP PCR through the detection of Pneumocystis colonisation of the respiratory tract. However, meta-analyses demonstrate excellent specificity, associated with positive likelihood ratios sufficient to confirm infection when positive Table 1. The PCP PCR positivity in upper respiratory tract samples (e.g. sputum) was once thought to reflect colonisation, but it is now believed to be specific for PCP disease, reflecting a significant burden in the lower respiratory tract of susceptible patients.
The overall performance of (1-3)-β-D-Glucan (BDG) when testing serum for the detection of IFD, other than PCP, as determined by meta-analysis generated sensitivity/specificity of 77%/85%, respectively. The sensitivity of BDG detection for the diagnosis of PCP is improved (>90%), but less so in the HIV-negative patient. On the basis of its high, negative predictive value, the National Institute for Clinical Excellence (NICE) has recommended that negative Fungitell BDG results are sufficient to exclude IFD.
A recent Cochrane review on the performance of assays for the detection of BDG demonstrated the following: Findings of Cochrane review: performance when diagnosing IFD in the ICU and cancer patients were similar, yet **significant variability** in both sensitivity (ICU range: 27-100%; Cancer range: 33-100%) and specificity (ICU range: 20-94%; Cancer range: 0-100%) was evident between studies. Performance for the detection of invasive candidiasis generated sensitivity/specificity of 81%/64%, respectively, with performance similar in neonates (Se/Sp: 89%/60%). Performance for the detection of BDG in relation to IFD in haematology patients was similar (Se/Sp: 80%/63%)
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Due to the presence of commensal Candida species and other potential fungal colonisers, BDG testing of respiratory samples is not recommended due to significant false positivity. All positive BDG results should be interpreted in clinical context and with an understanding of the potential specificity issues.

Combining multiple biomarkers enhances confidence when excluding infection, but conversely the presence of multiple positive biomarker results significantly improves specificity for confirming a diagnosis. For the diagnosis of IA various clinical trials have supported the combined used of GM-EIA and Aspergillus PCR, and a meta-analysis of this combined approach generated sensitivity and specificity of 99% and 98%, respectively.

By combining BDG serum testing with PCP PCR of upper respiratory tract samples, a diagnosis of PCP can be confidently achieved or excluded without the need to obtain invasive, deep respiratory samples. The combined strategy for the optimal diagnosis of IC is currently being determined by the A-STOP trial. To read more about this trial please click on the links in the see also section below.

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Fungal Diagnostics in Critically Ill Patients

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