Experimental models and clinical studies have shown PCR to be more sensitive than culture for detection of candidaemia. The sensitivity of the Candida PCR assay was 95.0% in one study, compared with a sensitivity of 75.0% for the Candida ELISA aiming to detect mannan (a difference not statistically different).¹⁰⁴ The specificities of the Candida PCR and ELISA were the same at 97.0%. In 45% of these patients, the PCR method detected the infection earlier than the ELISA. Newer realtime PCR assays such as TaqMan and the Light Cycler require no postamplification manipulations and can potentially be automated for all steps from DNA extraction to final PCR amplicon detection and quantitation.⁵⁹

It should be noted that there have been several headto- head comparisons of the various assays for detection of the Candida antigens discussed earlier. Unfortunately, none of the assays have performed well enough or has good enough predictive value at this point to be able to recommend its routine use in a clinical laboratory. Perhaps a combination of two assays may increase the accuracy of diagnosis of invasive candidosis.¹³⁰

Markers for Other Invasive Fungal Infections

The amplification of gene sequences unique to fungi is conceptually appealing, offering the potential for rapid and sensitive diagnosis of invasive fungal infections. In general, assays targeting multicopy genes have better detection limits than those targeting single copy genes.¹³⁵ Although PCR may become a diagnostic modality to identify Mucorales,^136–138 Scedosporium,^139–141 and several other fungi,^142–151 no commercial test is available to date. These techniques hold promise, but they are not yet standardised or readily available in most clinical laboratories. Also, large clinical trials to determine the sensitivity and specificity of such molecular tests are non-existent.

Role of the CT Scan

The ‘halo sign’ refers to a zone of ground-glass attenuation surrounding a pulmonary nodule or mass on computed tomography (CT) images.¹⁵² The presence of a halo of ground-glass attenuation is usually associated with haemorrhagic nodules.¹⁵³ In severely neutropaenic patients, this suggests infection by an angioinvasive fungus, most commonly Aspergillus. The halo sign is documented in 33% to 60% of patients with invasive aspergillosis and is short-lived.¹⁵⁴ To be useful for the diagnosis of aspergillosis, the CT scan must be performed within five days of the onset of infection, because ~75% of the initial halo signs disappear within a week.¹⁵⁵ The ‘air crescent’ sign does not appear until the third week of the illness, and its appearance may be too delayed to be helpful in the diagnosis of invasive aspergillosis.¹⁵⁴

Although usually regarded as an indication of haemorrhagic nodules, the halo sign may also be present when tumour or inflammatory cells infiltrate the lung parenchyma.^{153,155–159} The halo has been described in patients with eosinophilic pneumonia, bronchiolitis obliterans organising pneumonia,¹⁶⁰ and tuberculosis,¹⁶¹ and in patients infected by Mycobacterium avium complex,¹⁵⁵ Coxiella burnetti,¹⁶² cytomegalovirus, herpes simplex virus,¹⁵³ and myxovirus.¹⁶⁰ Patients with posttransplantation lymphoproliferative disorders ¹⁶³ and Wegener granulomatosis may develop the halo sign,¹⁵³ as well as some of those who have undergone transbronchial biopsy.¹⁵⁵ Although it is less common, the halo sign may also be observed in non-haemorrhagic nodules, in which case either tumour cells or inflammatory infiltration account for the halo of ground-glass attenuation.¹⁵³ Nonetheless, in the appropriate clinical setting, the halo sign is considered to be early evidence of pulmonary aspergillosis even before serologic tests become positive,¹⁶⁴ and it warrants the administration of systemic antifungal therapy.¹⁶⁵

Future Perspectives

Invasive fungal infections constitute a major challenge for the management of immunocompromised patients, mainly haemato-oncology patients, transplant and allo-HSCT recipients. In order to improve the survival for these infections, an early diagnosis is required. As shown in this article, conventional microbiological, histological and radiological techniques remain the cornerstone of diagnosis but are insensitive and have a limited impact on clinical decision-making. There is an urgent need for new and efficient diagnostic methods. These tests should be fast and highly sensitive. In addition, the recognition of the causal agent should be very precise. With the advance of molecular tools, new fungal species and new mechanisms of resistance will be clarified. DNAand RNA-based methods hold promise for improved sensitivity and specificity, but these methods will require extensive validation in clinical studies. Finally, costs are also very important in selecting an appropriate diagnostic test for invasive fungal infections.