Introduction

Chronic pulmonary aspergillosis (CPA) is a fungal infection disease characterised by cough, haemoptysis and weight loss present for at least for 3 months in non-immunocompromised people [1••]. This disease is caused by Aspergillus spp. that can reach the respiratory tract by airborne transmission [2]. Aspergillus spp. have been known as a cause of human infection for centuries. In 1842, Bennett observed fragments of ‘tubes’, somewhat larger, more or less matted together, which appeared distinctly jointed in microscopic examinations of sputa and the lining membrane of tubercular cavities in the lungs of a man initially thought to have tuberculosis [3]. Despite being first described > 100 years before invasive aspergillosis, more attention has been focussed on acute invasive aspergillosis in recent years, but now with the development of new (and welcome) antifungal agents, attention has refocused on CPA. The first report of amphotericin B use for any form of aspergillosis was for a case of CPA [4]. In the 1960s, most efforts related to aspergillosis were focussed on the therapy of invasive aspergillosis with amphotericin B and allergic aspergillosis. The one exception was a large Medical Research Council study in the UK published in 1968 which found an aspergilloma associated with Aspergillus precipitating antibody in 17% of pulmonary tuberculosis survivors who had residual lung cavities [5].

A watershed study in 1983 retrospectively compared medical and surgical treatment of patients with ‘pulmonary aspergilloma’. The authors suggested that medical therapy (which did not include antifungal agents) was not as effective as surgical resection [6]. A small study of low dose itraconazole (200 mg daily) published in 1991 was found to be ineffective [7], whereas the current recommended dose is 400 mg daily. These findings lead to medical passivity around CPA, and only surgical resection in those with resectable aspergillomas was offered for therapy. The publication in 2003 of a case series of 18 patients with full diagnostic criteria and antifungal therapy responses generated renewed interest in the problem by respiratory physicians [8]. The subsequent publication in 2011 of the magnitude of the problem after pulmonary tuberculosis globally (estimated at 1.17 million) has further focussed minds on addressing the problem [9].

CPA Burden of Disease

CPA cases after tuberculosis can be found worldwide, from an estimated 11,400 cases in Europe to 145,372 cases in South-East Asia in 2007 and these numbers are likely to keep increasing each year [9]. In most countries, the number of CPA cases correlates with the number of tuberculosis (TB) patients because the fungus can colonise and infect cavities left in the lungs after TB infection [5, 10]. There are an estimated 1,170,000 cases of CPA after TB and at least the same number or more cases from other pulmonary disorders namely chronic obstructive pulmonary disease (COPD), sarcoidosis, non-tuberculous mycobacterial infection (NTM), post-pneumothorax, allergic bronchopulmonary aspergillosis (ABPA) and rheumatoid arthritis [9, 11]. Untreated CPA has a high mortality rate, causing around 450,000 deaths every year [12]. Several factors are associated with a higher mortality risk for CPA including NTM infection, COPD, pleural involvement, cavitary disease, presence of an aspergilloma, shortness of breath, low physical activity, and low body mass. Those with multiple pulmonary infections such as NTM infection may be linked to subtle immunodeficiency, such as poor vaccine response rates and abnormally low CD4, CD19, and CD56 (natural killer) cell counts, as well as either gamma interferon or interleukin 12 deficiency [13,14,15]. The 1-, 5- and 10-year survival rates are 86%, 62% and 47%, respectively [16•].

CPA can manifest in different forms, which are sometimes overlapping (Table 1). Some patients will be diagnosed with only a single fungal ball or aspergilloma. CPA can also appear as one or more nodules in the lung although this is unusual. Other patients can have one or pulmonary cavities that can develop extensive lobar fibrosis over weeks or months after chronic fibrosing pulmonary aspergillosis (CFPA). Two entities that can precede CPA include community acquired pulmonary aspergillosis and subacute invasive pulmonary aspergillosis in moderately immunocompromised patients [17].

Table 1 Description of different forms of CPA [1••, 17, 18]
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Diagnosis of CPA

The diagnosis of CPA is made with a combination of clinical symptoms (recognising that the early phases of the disease are often asymptomatic or clinically subtle), characteristic radiological findings and laboratory tests indicating infection with Aspergillus spp. [1••]. To distinguish the disease from sub-acute invasive aspergillosis, an arbitray duration of at least 3 months is required, and no, or only subtle, immunocompromise. With respect to corticosteroids, we usually take < 10 mg of prednisolone long term or equivalent as that threshold to define immunocompromise, but again this is arbitrary. Acute invasive, sub-acute invasive and community acquired Aspergillus pneumonia can all persist beyond 3 months and sometimes it it helpful to re-categorise such patients as having CPA in terms of ongoing management [1••, 17]. Besides diagnosing CPA, co-infection such as NTM/TB (these infections can occur concurrently), other bacterial infections and endemic fungal infections including histoplasmosis and coccidioidomycosis should be excluded [16•, 19].

Besides the main diagnostic criteria for CPA, there are other findings that can distinguish other disorders from CPA. Chest radiographs remain the first imaging modality for the suspicion and diagnosis of CPA, but CT of the thorax offers much additional value, especially in identifying aspergillomas. Chest radiographs without cavitation or pleural thickening had a 100% negative predictive value for chronic cavitary pulmonary aspergillosis in the African setting [20••], such work has yet to be done rigorously in high-income countries. The imaging features of CPA result from a combination of the findings related to underlying lung disorders and changes secondary to Aspergillus infection itself, reflecting the chronic inflammatory and immune response to Aspergillus spp. [1••]. The presence of anti-Aspergillus antibodies differentiates between infected and colonised patients with a positive predictive value of 100% for detecting infection, but it does not distinguish which form of aspergillosis it is.

Clinical Symptoms

Aspergillosis is generally clinically silent with symptoms appearing late in the course of disease. There are many clinical symptoms that can be found in CPA patients such as weight loss, productive cough, haemoptysis, fever, sputum production, fatigue and shortness of breath. Fever is unusual and may suggest SAIA (subacute invasive aspergillosis) or an alternative infection such as tuberculosis. CPA patients must have one or more symptoms with a duration at least for 3 months and absence of overt immunosuppression [2, 8, 21, 22]. These symptoms are not specific, hence the need for additional data to confirm or refute the diagnosis. Some patients have very few symptoms but do have progressive disease, so reliance on symptoms alone to decide on therapy is sub-optimal clinical practice.

Radiological Findings

The unique radiological appearance that Deve described in 1938 as ‘mycotic lung tumor’ or what we now called a fungal ball or aspergilloma [23] is highly distinctive. Aspergilloma usually can be found in upper lobe, with air crescent and pleural thickening around it (see Fig. 1). Radiological features of CPA vary, but most authorities require at least one of two radiological appearances [1••]. First is a cavitary lesion with paracavitary fibrosis. Second is a new or expanding cavity on serial imaging [8, 22]. One or more pulmonary cavities with paracavitary infiltrates, including pleural thickening or parenchymal infiltrates, were the most common CT abnormalities detected [22]. Progression was usually characterised by coalescence of cavities to form larger ones, or fibrosis [8].

Fig. 1
figure 1

a An example of a left upper lobe cavity containing a fungal ball (aspergilloma) with an air crescent above it (1:00 to 3:00) and pleural thickening together with some other parenchymal infiltrates separate from the area of aspergillosis. b Chest x-ray from same patient in panel a, the fungal ball has gone (it was coughed up) leaving only pleural thickening and slightly more peri-cavitary infiltrate infero-laterally, most consistent with progressive CPA, even though the fungal ball has gone. c Typical appearance of CCPA on CT scan with a thick walled cavity in the right upper lobe, probably containing a very small amount of fungal material laterally with marked pleural thickening, and an area of cavitating consolidation posteriorly, on a background of marked emphysema. d The corresponding PET scan of panel c that shows how much inflammation there is in the areas of pleural thickening and consolidation, showing up the small area of cavitation within the area of consolidation. (source: National Aspergillosis Centre)

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There are other manifestations of CPA that present as single or multiple nodule(s) without cavitation; this presentation can be missed as these findings are often associated with negative Aspergillus IgG or precipitins, or no biopsy to secure the diagnosis. Growing usage of computed tomography (CT) scanning has increased the chance of finding non-calcified pulmonary nodules, especially with lung cancer screening [24]. Many nodules found on lung cancer screening are resected or if impossible will be treated with radiotherapy which is not ideal for CPA [25••]. CCPA if left untreated may progress to CFPA. The appearance is no different from other forms of fibrosis but cavitation and fungal balls may be seen around the areas of fibrosis, and progression over time may provide a clue to the aetiology of the fibrosis.

Laboratory Investigations

Confirmation of CPA by laboratory testing normally consists of either detecting an elevated Aspergillus precipitin titre or an abnormally high Aspergillus IgG level, the precise cutoff depending on the actual test used. Conidia of Aspergillus are covered in rodlet proteins which are ‘invisible’ to the immune system. Once the conidia swell and germinate, multiple antigenic proteins are released and generate inflammatory responses, including dendritic cell and T cell activation [26]. The cell wall of A. fumigatus is mainly composed of polysaccharides, with many linked to proteins as glycoproteins. The majority of the antigens secreted by the fungus during its in vitro and in vivo growth are in or adherent to the cell wall and stimulate antibody production. [27].

Serological Tests

Immunodiffusion (ID) is one of the earliest methods to detect antibodies in serum samples [28], also known as agar gel double diffusion, precipitation in gel or the precipitin test. The principle of this test is the formation of an antigen-antibody complex that is visible in an agar gel. As the name implies, both antigen and antibody diffuse through the gel. It is used extensively to check antisera for the presence and specificity of antibodies to a particular antigen or antigens [29]. It was first used in the diagnosis of CPA in 1964 when the presence of specific precipitins in patients’ serum against antigenic extracts of cultures of A. fumigatus were considered to have value in the diagnosis of pulmonary aspergillosis [30]. Although no complex equipment is needed, the method takes a couple of days to perform, is labour intensive and relies on subjective interpretation of results [31].

Counterimmunoelectrophoresis (CIE) is a modification of immunodiffusion. This method produce precipitin lines faster than immunodiffusion because it applies electrical fields to make the movement process of antigen-antibody faster [32]. CIE is preferred over ID in most laboratories not only for its increase rapidity but also because it can reduce the amount of reference sera and antigen extract needed for testing [33]. Although the sensitivity of CIE is similar to ID [34], some laboratories still use ID even now, as it has been an established method for > 40 years. This is because there are a number of troublesome disadvantages of CIE. Since reagents migrate as finite zones, with essentially no concentration gradients, precipitins will form only if antigen:antibody equivalence becomes established in the area of the gel between the wells. Virtually, no time is available for second-stage antigen:antibody complexes to accumulate. Nowadays, both assays are less sensitive than other antibody detection methods and slower.

Enzyme-linked immunosorbent assay (ELISA) has been used to detect antibody in aspergillosis patients for decades [35]. There are several different types of ELISA, but the most commonly known are competitive and sandwich type. In competitive ELISA, labelled antigen competes with unlabelled antigen for binding to a limited quantity of antibody. In sandwich ELISA, antigen reacts with excess solid-phase antibody, and the bound antigen is treated with excess labelled antibody [36].

Most ELISA for CPA diagnosis must be performed manually, but it can be fully automated too [37, 38, 39•]. Bio-Rad (Marnes-la-Coquette, France) and Bordier Affinity Products SA (Crissier, Switzerland) are examples of diagnostics companies that produce ELISA assays for detecting Aspergillus antibody IgG. Phadia ImmunoCAP (Thermo Fisher Scientific Inc., Uppsala, Sweden) and Immulite (Siemens AG, Munich, Germany) are an automated variant of ELISA that being used in many large hospital laboratories. Both of them use a similar principle where Aspergillus antigen that is coupled to the solid phase will react with specific IgG antibodies in the patient sample. Enzyme labelled antibodies against IgG are added after washing away non-specific IgG antibodies. The bound complex formed is then incubated with a developing agent before stopping the reaction and measurement of fluorescence [37]. The performance of ELISA tests that already available commercially are varied, but the recommended assay preferably have 90% sensitivity and 85% specificity [40]. The sensitivity and specificity of the Bio-Rad, Bordier, ImmunoCAP and Immulite were 86–97.4% and 89.6–98.2%, 97.4 and 90.3%, 83.8–97.9% and 98%, 92.9–96% and 98–99.3%, respectively [37, 38, 39•, 41,42,43].

Lateral Flow Device

Lateral flow devices (LFD) are inexpensive, simple to use disposable devices that can identify a substance in a clinical sample effortlessly. It has developed from the principle of latex agglutination assays that were used as a test to diagnose rheumatoid arthritis more than half century ago [44]. Serological lateral flow tests then emerged 30 years ago [45]. A good example is the well-known human pregnancy LFD that was derived from the development of a hCG beta–subunit radioimmunoassay [46]. It did not take a long time for commercial rapid lateral flow tests to be used in other fields besides clinical diagnostics particularly in veterinary, food, environmental applications, bio-defense and drug abuse [47].

LFD strips contain four main components as shown in Fig. 2. The sample application pad is the location where the sample is applied. It is made of cellulose and/or glass fibre. The conjugate pad is the place where labelled biorecognition molecules are dispensed. The nitrocellulose membrane is where test and control lines are located. An adsorbent pad is at the end of the strip that maintains the flow rate of the liquid and stops backflow of the sample [48]. An LFD test is now available for Aspergillus IgG detection.

Fig. 2
figure 2

Schematic diagram of lateral flow device strip. Sample will move from sample pad to adsorbent pad. The direct format gives colour appearance at both of test and control lines when a sample with target analytes is applied

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The principle is to let sample flow in one direction which will bind to test line if it contains the protein of interest and the result can be interpreted with a naked eye. Some tests have a reader which is slightly more sensitive and allows the result to be captured for laboratory reporting using format reporting procedures. The only commercially available lateral flow assay available for detecting Aspergillus-specific antibodies currently is the LDBio Aspergillus ICT lateral flow assay. This assay has 88.9–91.6% sensitivity and 96.3–98% specificity that is comparable with ELISA tests that already being used widely above [49•, 50•]. This assay has not been used quantitatively.

Serology Test in Resource-Constrained Settings

Based on the epidemiology data, most of CPA cases are most probably located in low-middle income countries [9]. The current guideline for the diagnosis of CPA includes Aspergillus IgG detection to prove microbiological evidence of Aspergillus spp. infection in resource constrained settings [40]. Although many serology tests for CPA available now, not all healthcare systems have access to them [51]. As described before, many CPA cases in resource-limited settings are left untreated because of the lack of diagnostics. The most suitable solution for this problem is lateral flow device as point-of-care test. Lateral flow device meets the World Health Organization’s ASSURED criteria for being affordable, sensitive, specific, user-friendly, rapid and robust, equipment free, and deliverable to end-users [52].

Other Tests

Direct microscopy is a well-established diagnostic test that is still used today to identify fungi, especially filamentous fungi in clinical samples. This method is used for fungal diagnosis because the morphology of many fungi is quite different. The presence of fungi in samples is usually determined by direct microscopy of the clinical specimen on a microscopic glass slide with 10% of potassium hydroxide (KOH) for wet mount examination or enhanced by brightener agent like Calcoflour white. Direct microscopy in the diagnosis CPA can help the laboratory to decide which culture media and procedures to use if fungal hyphae are seen on sample [53].

Fungal culture and identification can detect fungi from many kinds of samples with low to medium sensitivity for all fungi (except Pneumocystis and Microsporidia which are not readily culturable), and have 3–14 days turnaround time [54]. Aspergillus species are ubiquitous saprophytic fungi that are distributed widely in the environment. From hundreds of known species, less than 40 cause disease in humans. The most common pathogens are A. fumigatus, A. flavus, A. terreus, and A. niger which are also the most commonly reported species of Aspergillus in nature [55]. The most common cause of CPA is A. fumigatus, although A. flavus and A. niger also can cause CPA [2, 56]. The presence of A. fumigatus in a bronchoscopic specimen is far more common in infection compared to colonisation and is consistent with infection, including CPA. A recent study showed that high volume culture has good sensitivity compared to conventional culture; it also increase the recovery of cryptic species from sample [57, 58].

Polymerase chain reaction (PCR) is a molecular method that has a higher sensitivity since it allows the detection of small amounts of deoxyribonucleic acid (DNA) in clinical samples by amplification. Extraction of DNA from hyphae and/or conidia is an important step for the detection of fungi using PCR. The structure of the fungal cell wall is highly complex and no single universal fungal DNA extraction method has been adopted [59]. There are various studies describing PCR for the detection of Aspergillus spp. in the context of CPA; the average sensitivity and specificity is around 80% [60, 61, 62•, 63].

Differential Diagnosis of Positive Aspergillus Antibody and Aspergillosis Syndromes

Invasive aspergillosis is a life-threatening disease that commonly occurs mostly in immunocompromised person, such as people with acute leukaemia, stem cell and other transplants (especially lung) [64, 65]. Aspergillus IgG antibody can be detected in invasive aspergillosis although it is less important than Aspergillus antigen detection [65, 66]. Aspergillus bronchitis is chronic superficial infection of the lower airways (characterised by bronchial cast containing mucus and mycelia of any Aspergillus pathogenic species) that commonly occurred in non-immunocompromised patients. Aspergillus-specific IgG may be positive or negative in Aspergillus bronchitis cases [67]. Aspergillus rhinosinusitis characterised by a mass within one or more paranasal sinuses with evidence of invasion of contiguous structures such as the base of the skull, orbit and brain [68]. Species specific precipitating antibodies are present in > 90% of cases in chronic invasive disease and correlates well with disease progression, usually to A. flavus [69, 70]. Similarly, precipitating antibodies to Aspergillus spp. are present in around two-thirds of cases of chronic granulomatous sinusitis, especially a positive IgG to A. flavus [71,72,73]. Chronic pulmonary histoplasmosis presents in a similar way to CPA with cough, weight loss, fever, chest pain, fatigue and haemoptysis the most frequent symptoms in decreasing order of frequency. Histoplasma antibody is usually detectable although there is possible cross-reaction with Aspergillus (assuming not a dual infection) [19, 74]. Coccidioidomycosis may present with acute pneumonia, nodules or chronic cavitation. Sputum culture is usually positive in cavitary disease. Anti-coccidioidal antibody detection is important for diagnosis with probably little cross-reaction with Aspergillus [75,76,77]. Fungus ball as a result of superinfection with other fungi including Aspergillus sp. can be formed when cavities are present.

Conclusion

CPA has been neglected for a long time despite its high morbidity and mortality, because it has similar clinical symptoms and radiological appearance to other lung diseases. Laboratory diagnostics, especially Aspergillus-specific IgG assays are very valuable in the diagnosis of CPA. There are many different options of serological tests available, including ELISA, with differing performance characteristics [37]. The ELISA capability to measure IgG titre quantitatively is important to monitor CPA progress and treatment [78, 79]. The lateral flow assay is an option as a simple diagnostic test for CPA, particularly in a resource-constrained setting. It is inexpensive and simple to run and accessible to many users. The current lateral flow assay for the detection Aspergillus-specific IgG that are commercially available can only produce a qualitative result, but it has potency to become a semi-quantitative or quantitative test [80, 81]. We hope that the availability of new tests will translate into the earlier diagnosis of CPA allowing clinicians to start treatment earlier resulting in a better prognosis. People from resource-constrained settings where probably have higher CPA prevalence will get more benefit from point-of-care tests such as a lateral flow device.