Target condition being diagnosed

Invasive aspergillosis (IA) is a disease resulting from opportunistic fungal infection and mainly affects immunocompromised hosts, particularly neutropenic patients such as those undergoing cancer treatment and hematopoietic stem cell transplantation (HCT) and solid organ transplant recipients (Flückiger 2006; Marr 2002). The highest incidence (10% to 20%) and mortality rates (60% to 90%) of IA have been reported following allogeneic HCT and heart, lung or heart/lung transplantation. The principal reason for such people developing IA is that the underlying disease and its subsequent treatment with chemotherapy induces bone marrow failure resulting in profound leucopenia and impaired innate and cell‐mediated immunity. The leucopenia is marked by a lack of functioning polymorphonuclear leucocytes, resulting in the patient lacking the phagocytic white blood cells needed to fight infections, including aspergillosis. Innate immunity is further impaired by iatrogenic damage to the local defences of the oral cavity, gastrointestinal tract and respiratory tract. Damage to the respiratory tract is poorly understood but hampers effective clearance of fungal spores, especially those of Aspergillus fumigatus which are ubiquitous in the environment, readily airborne and small enough to lodge in the alveolar spaces. The lack of local and systemic immune defences means that any spores that germinate can infect lung tissue and progress to a full‐blown infection. The disease that follows is characterised by invasion of the capillaries (angioinvasion) which can lead to further dissemination to other parts of the lung and indeed other organs, particularly the brain.

Early diagnosis of IA and prompt administration of appropriate antifungal treatment have been recognised as crucial to the survival of people with IA (Marr 2002; Walsh 2008). Antifungal drugs can be given for prevention of infection (prophylaxis), treatment of unexplained fever (empirical therapy), treatment of non‐specific clinical features or mycological evidence (pre‐emptive therapy) and treatment of possible, probable and proven invasive fungal disease (IFD) (directed therapy). Clearly, the earlier that treatment is started the better the outcome. Consequently there is an urgent need for new diagnostic tools to detect infection before disease becomes manifest, thereby allowing effective treatment strategies to be developed. The polymerase chain reaction (PCR) is becoming increasingly popular (Arvanitis 2014; Donnelly 2006; Hope 2005; Mengoli 2009; Tuon 2007; White 2015). However it was not considered robust enough to be included in the international consensus definitions of the European Organisation for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG); (Ascioglou 2002; De Pauw 2008).

The prevalence of IA varies from 1 in 100 to about 1 in 6 depending upon the level of compromised immunity, the environmental exposure and preventative measures taken, which can include protected isolation with filtered air and antifungal prophylaxis. The outcome depends upon the extent of infection, whether diagnosis is made and treatment with an effective drug is initiated early; and, importantly, whether or not an individual's immune system begins to recover (Marr 2002; Walsh 2008).

Demonstration of fungi in diseased tissue is still required for a proven diagnosis of IFD. Unlike other infectious diseases, direct demonstration of Aspergillus infection is rarely possible by culture of sterile body fluids, and obtaining tissue from a live patient is seldom feasible because of the risks posed to the patient.by biopsy.

Recently, advances have been made on several fronts. The EORTC/MSG's published definitions of invasive fungal disease (IFD) allow for degrees of certainty of diagnosis: possible, probable and proven (Ascioglou 2002; De Pauw 2008). Definitions of invasive fungal infection were devised in 2002 and revised in 2008 to focus on fungal disease (Table 1). These are based on host factors, radiological features and mycological evidence. Probable and possible cases have to satisfy the same host and radiological criteria and they are only distinguished by the presence or absence of mycological evidence. Biomarkers have potential to detect infection before development of overt disease, allowing treatment to be initiated at an earlier stage. These definitions were only made possible by other contemporaneous developments in the field. Computer‐assisted tomography (CT scan) became more widely available, allowing lesions consistent with pulmonary IA to be detected at an early stage of disease. This offered the possibility of performing bronchoscopy to obtain bronchoalveolar fluid in which the fungus could be detected by microscopy and culture as well as galactomannan (GM). However the technique is not without risk and cannot always be performed when required. By contrast, blood is readily available which opens up the possibility of looking for fungi in an indirect fashion by detecting fungal cell components including the galactomannan of the cell wall of Aspergillus species (Leeflang 2008).

The EORTC/MSG definitions help integrate all the clinical and laboratory information available. Combining of host factor (such as neutropenia) with clinical features (such as pulmonary nodules) and mycological evidence (such as detection of GM) allows a high level of certainty of diagnosis to be assigned. These definitions have been adopted widely by regulatory agencies, such as the European Medicines Agency and the US Food and Drug Administration, for evaluating antifungal drug products and diagnostic tests, as well as by the scientific and medical community at large for investigating epidemiology and auditing antifungal stewardship.

Whilst the range of potential drugs currently available allows prophylaxis, pre‐emptive therapy, as well as directed therapy for possible, probable and proven IFD, the ability to identify 'who needs treatment, when, and with what' is sufficiently unreliable that many physicians continue to treat empirically. Not only does this lead to unnecessary costs but it is also not clear how many people are helped or harmed by this approach. There are circumstances when a host factor is present (for instance receipt of an allogeneic hematopoietic stem cell transplant (HSCT)) and mycological evidence exists (such as Aspergillus being recovered from pulmonary secretions) without evidence of active disease. This may represent infection before disease becomes manifest and provides the opportunity for therapy to pre‐empt disease. Consequently there is an urgent need for new diagnostic tools and an assessment of their utility in the clinic. Biomarkers have the potential to detect infection before development of overt disease, allowing treatment to be initiated at an earlier stage.

Index test(s)

There are few direct diagnostic tests and those that are available are limited by the difficulties in obtaining tissue specimens to allow culture, microscopy and histology (Chamilos 2006). Blood in its various forms — whole blood, plasma and serum — is readily available, but only tests for antigens such as GM and beta‐D‐glucan have been deemed acceptable to support a diagnosis (Leeflang 2008; Pfeiffer 2006; Senn 2008). In neutropenic patients, pulmonary abnormalities consistent with invasive aspergillosis, such as nodules, often surrounded by a 'halo sign', can be detected using high‐resolution computed tomography (Greene 2007). However, the 'halo sign' is transient and only detectable during early invasive aspergillosis, after which radiological signs become non‐specific or appear too late to be therapeutically useful (Caillot 2001). Radiological signs also herald established disease so the opportunity to intervene early has been lost.

Molecular methods, such as the PCR, have been investigated in order to improve the diagnosis of IA (Donnelly 2006; Mengoli 2009; White 2010; White 2015). PCR can amplify a single or a few copies of target DNA allowing target detection with great sensitivity and specificity. Moreover it can be quantitative, using the procedural variant called real‐time PCR (qPCR). The sensitivity is based on the enormous potential for exponential amplification of the DNA target (the 'amplicon') due to repeated cycles of the polymerase reaction, where every cycle doubles the quantity of amplicon. Real‐time PCR continuously monitors the amplification of target DNA at every cycle. The threshold cycle number (preferred term Cq) is when the amplicon becomes detectable above the background level, as an exponentially increasing signal, and is proportional to the amount of starting DNA in the reaction. A high initial DNA concentration will require fewer cycles to reach the threshold and has an earlier Cq value. The specificity of PCR resides in the DNA oligonucleotides used as primers, allowing the terminally stable variant of the enzyme DNA polymerase to initiate sequence duplication. These primers join to the DNA target ('annealing') in a very stringent way, allowing only minimal misfit possibility. Moreover, in quantitative real time PCR (q‐RT‐PCR), the use of reporter probes, hydrolysis probes or molecular beacons that bind to the central part of the target sequence increase the assay’s specificity.

PCR has an enormous potential for diagnosing infectious diseases, particularly where traditional culture methods are less effective. The fungal genus Aspergillus is a good example of this kind of approach. The recovery of Aspergillus from blood cultures is rarely achieved even in overwhelming infection. PCR‐based tests on blood specimens have gained popularity as the platforms become more automated and extraction methods and targets become commercially available (White 2010). However, its exclusion from the EORTC/MSG definitions led to the establishment of the European Aspergillus PCR Initiative (EAPCRI), which is a working group of the International Society of Human and Animal Mycology (ISHAM). The EAPCRI has published various studies describing the critical stages in DNA isolation from blood samples (White 2010), and on the critical characteristics of a standardized Aspergillus PCR assay. These studies, allied to the standardization of qPCR assays described in the MIQE (minimum information for publication of quantitative real‐time PCR experiments) guidelines, have helped pave the way for reliable and robust PCR assays for the diagnosis of IA in the clinical setting (Bustin 2009). Progress in the standardization of methodology enabled the development of commercially available Aspergillus PCR assays in the last few years (Rath 2018).

Clinical pathway

As stated above, many physicians still opt for starting antifungal treatment empirically because of diagnostic uncertainty. This approach can lead to unnecessary treatment, which incurs extra costs, and may be harmful to some people. Diagnostic tests can be used to establish (i.e. rule in or rule out) disease. This is particularly useful for people at risk of IA where a highly sensitive test can deliver a high negative predictive value for disease, allowing empirical therapy to be safely withheld even on the basis of a single test result. Conversely, a high positive predictive value is required to rule in the diagnosis. The use of PCR as a screening tool differs fundamentally from its use as confirmation of the diagnosis. Therefore, if prevalence is low (i.e. < 10%), IA can be ruled out during the risk period for as long as any single PCR test result is negative, and there are no clinical signs of disease. Conversely, two or more PCR positive test results could be used for mycological confirmation of clinically suspected disease, also allowing a case of possible IA to be upgraded to probable IA.

Clinical pathways of managing patients can vary according to the risk of IA. Patients at high risk can be screened using GM, PCR (or both), with positive test results being used to trigger an intensive diagnostic workup with CT scanning and bronchoalveolar lavage (BAL) to determine disease (diagnostic driven) or to initiate antifungal treatment to prevent development of disease (pre‐emptive). Screening may occur throughout the period of risk or only when people develop fever. Alternatively, patients may be tested in the presence of symptoms suggestive of disease to confirm diagnosis.

Rationale

There is no single assay that has been validated for the early diagnosis of IA. Non‐culture‐based methods such as serial GM ELISA screening hold most promise in establishing early diagnosis and may result in improved outcomes, but clinical utility has not been fully established. Moreover, newer methods such as PCR are being investigated (Donnelly 2006; White 2015). As with any diagnostic test, the utility of PCR as either a screening tool or a confirmatory test will depend on the prevalence of disease in the population in which it is used. The use of prophylactic or empirical antifungal agents, availability of protective environments and other diagnostic tests will all influence how the test is used in clinical practice. It is not the aim of this present analysis to establish clinical outcomes but rather to evaluate diagnostic accuracy so that rational use of PCR testing can be applied to different populations.