Abstract
In people with cystic fibrosis (PwCF), viscous sputum and dysfunction of the mucociliary escalator leads to early and chronic infections. The prevalence of Aspergillus fumigatus in sputum is high in PwCF and the contribution of A. fumigatus to the progression of structural lung disease has been reported. However, overall, relatively little is known about the contribution of A. fumigatus to CF lung disease. More knowledge is needed to aid clinical decisions on whether to start antifungal treatment. In this review, we give an overview of A. fumigatus colonisation and infection in PwCF and the different types of pulmonary disease caused by it. Furthermore, we discuss the current evidence for structural lung damage associated with A. fumigatus in PwCF on chest computed tomography and magnetic resonance imaging. We conclude that radiological outcomes to identify disease caused by A. fumigatus can be important for clinical studies and management.
Abstract
Specific structural lung damage on chest CT and MRI associated with A. fumigatus infection in CF can be identified. A. fumigatus-related structural lung damage can be detected on images at an early stage to guide treatment and clinical management in CF. https://bit.ly/3CWzQWq
Introduction
Cystic fibrosis (CF) is a multi-system disease caused by a mutation in the gene that encodes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein [1–3]. The CFTR protein defect leads to an abnormal composition of the epithelial lining fluid, viscous sputum and dysfunction of the mucociliary escalator [3–5]. As a result, most people with CF (PwCF) suffer from early and chronic airway infections caused by a wide range of microorganisms, especially Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus fumigatus [6–10].
There is a huge amount of knowledge available about bacterial infections in PwCF, but relatively little is known about the contribution of A. fumigatus infection. The prevalence of A. fumigatus-positive sputum cultures in adult PwCF ranges from 27% to 57% [11]. The prevalence of A. fumigatus in paediatric PwCF was thought to be relatively low under the age of 8 years, and more common in children older than 10 years [12]. However, the incidence may be significantly underestimated in children [13]. In 2018 and 2019, studies using bronchoalveolar lavage (BAL) samples taken from children up to the age of 6 years found the median age of colonisation with Aspergillus species (mainly A. fumigatus) in PwCF to be 3.2 years [14], and A. fumigatus to be present in up to 28% of patients from the age of 3 years onwards [15].
Previous research has indicated that A. fumigatus is not an innocent bystander but can contribute substantially to structural lung damage [11, 15–23]. However, it is not clear how to distinguish A. fumigatus-related lung damage from lung damage caused by other CF-related microorganisms. Diagnostic imaging techniques can be important to make this distinction and to aid clinical decision-making on whether to start antifungal treatment. The threshold to start such treatment in PwCF with positive cultures for A. fumigatus is high, as antifungal drugs can have substantial side effects. Furthermore, there are no well-conducted trials available in CF to guide treatment and anti-fungal drugs are known to have a high number of drug–drug interactions with other CF medications. Therefore, it is important to identify within the larger group of A. fumigatus-positive PwCF those individuals that require antifungal treatment to avoid accelerated progression of structural lung damage and lung function decline. For those subjects, negative consequences of antifungal treatment will likely be outweighed by the advantages, because halting or slowing down the progression of structural lung damage will have a direct effect on their life expectancy and quality of life.
For this review, we will first give an overview on how A. fumigatus colonisation and infection in PwCF can develop and discuss the different types of pulmonary disease caused by A. fumigatus. Next, we will elaborate on the role imaging can play in the detection of A. fumigatus-related structural lung damage and in the differentiation between the different types of pulmonary disease it can cause in PwCF.
Background of A. fumigatus
Sources of Aspergillus
Although over 150 species of Aspergillus have been described, only a few can cause disease in humans [24]. The most important Aspergillus species associated with pulmonary disease is A. fumigatus [20, 25]. Commonly, A. fumigatus grows ubiquitously in soil, on carbon-rich substrates or as a contaminant of starchy foods such as bread and potatoes (figure 1a). Furthermore, studies have shown that patients colonised with A. fumigatus may become the source of patient-to-patient transmission via cough aerosols or sputum [26, 27]. A. fumigatus has different stages in its life cycle with different morphological forms. Conidia, which are tiny hydrophobic particles, are easily dispersed by air and are inhaled on a daily basis by humans (figure 1b). Conidia are the infective form of the Aspergillus, while hyphae are the invasive form causing invasive disease (figure 1c, d). Conidia in the airways of PwCF can become metabolically active and secrete antigens which can provoke an allergic response. Once conidia land in a suitable environment with sufficient nutrients, such as the lungs, they can start to germinate and form hyphae which can cause an invasive infection (figure 1d) [28, 29].
Inhalation
People inhale on average between 100 and 1000 conidia per day [28, 30, 31]. The mass median aerodynamic diameter of the conidia is in the order of 2 to 3 µm, with extremes up to 3.5 µm [31, 32]. Hence, conidia are in the respirable range, meaning that they are small enough to bypass the upper airways to get deposited into the peripheral airways and even into the alveoli [33–35]. In healthy subjects, mucociliary clearance will be able to clear inhaled conidia effectively. Furthermore, alveolar macrophages as the first line of defence can eliminate conidia at an early stage, thereby avoiding germination [11, 16, 36–38]. In PwCF, the mucociliary clearance is severely compromised and CFTR-defective alveolar macrophages are less effective in the elimination of conidia. Therefore, inhaled conidia have a higher chance to germinate. Furthermore, it has been shown that dysfunction of epithelial cells in CF influences the interaction with A. fumigatus and leads to inflammation of the lungs [16, 39].
Colonisation
Colonisation by A. fumigatus is defined as the presence of this fungus in laboratory cultures of the respiratory tract without clinical symptoms or deterioration in lung function (table 1 [13, 40, 41]) [16, 25, 42]. The exact prevalence of colonisation in PwCF is not well known and reports vary at between 3.4% and 57% [11, 38, 43].
Rates of colonisation are associated with levels of exposure [11, 44]. Certain climate factors, such as temperature, wind speed and humidity alter the load of A. fumigatus in the air [45], which may influence rates of colonisation in PwCF. For example, most reports show that higher concentrations of airborne conidia are detected in warmer weather, especially during summer and autumn, but without significant differences both in colonisation rate and airborne counts [46–49].
Risk factors for A. fumigatus colonisation include previous treatment with steroids or antibiotics. Short courses of steroids in PwCF have been associated with an increased risk of positive sputum cultures for A. fumigatus [11, 13, 20, 25, 34, 50–55]. Furthermore, long-term antibiotic exposure may also provide a more favourable environment for fungi to grow, causing A. fumigatus colonisation to occur [6, 54, 55].
Interactions
Another risk factor for persistent colonisation with A. fumigatus is co-infection with P. aeruginosa. There is a lot of conflicting literature about the interaction of co-infection between A. fumigatus and P. aeruginosa. The prevalence of chronic P. aeruginosa infection currently ranges from 3.3% to 26% for children and between 41% and 52% for adults with CF [56]. In vitro research has shown that the production of cytotoxic elastase by P. aeruginosa increases in co-culture with A. fumigatus, leading to damage to the human lung epithelial cells [54, 57]. Alternatively, eradication treatment for P. aeruginosa could create a microbial environment favourable for A. fumigatus colonisation [58]. Data from the Irish CF registry showed that co-colonisation with P. aeruginosa and A. fumigatus was associated with a 165% increase in hospital admissions, a 112% increase in the number of respiratory exacerbations and a 48% increase in the number of oral antimicrobial courses, compared to patients with negative cultures for both pathogens [59].
Apart from P. aeruginosa, interactions between A. fumigatus and other bacterial infections, such as Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae and Stenotrophomonas maltophilia, do not only lead to superinfection but also increase the chance of A. fumigatus infection evolving into pulmonary disease in PwCF [60]. In addition, in PwCF, a positive fungal culture is often associated with a positive culture for nontuberculous mycobacteria [61, 62], a combination that seems to predispose to the development of chronic pulmonary aspergillosis [63, 64]. Therefore, further research is needed on these complex interactions between microorganisms and their effect on clinical outcomes in PwCF.
Recently, the interaction between anti-fungal treatment and CFTR modulators has attracted attention in PwCF, as the treatment of chronic Aspergillus infection has become troublesome for those who are also receiving modulator therapy. CFTR modulators, which can enhance the functional expression of specific CFTR mutations, also impact fungal colonisation [65]. On one hand, CFTR modulator studies have shown ivacaftor, lumacaftor and tezacaftor to have a positive effect on the dysregulated host response to Aspergillus in PwCF [66], which may reduce airway inflammation from Aspergillus infection. On the other hand, CFTR modulators can have drug–drug interactions with antifungal medication. For instance, the cytochrome P450-3A-inducing effect of lumacaftor can make it difficult to reach clinically effective blood levels of orally administered antifungal medications [65].
Infection
A. fumigatus infection occurs when the conidia in the airway trigger the release of harmful factors such as antigenic proteins and toxins, and/or germinate into hyphae becoming invasive (figure 1d), thereby initiating an inflammatory response. Neutrophils are recruited resulting in high levels of reactive oxygen species which mediate a multitude of inflammatory pathways causing even more damage to the airways [67, 68]. Furthermore, A. fumigatus produces some secondary metabolites, such as proteolytic enzymes and gliotoxins, leading to epithelial cell detachment and further impaired mucociliary clearance and enhanced inflammation [6, 18, 34, 35, 38]. A mycobiome analysis approach in a non-CF bronchiectasis cohort has been reported, which stated that the pulmonary mycobiome, in which A. fumigatus is one of the dominated profiles, is of clinical relevance in the development of bronchiectasis [23]. The multi-omics approach will likely be a benefit for diagnosing Aspergillus infection, as the characterisation of the host–fungal interaction and the use of multi-omics strategies may lead to a better understanding of host biology and its interaction with fungal pathogens [69].
In PwCF, pulmonary aspergillosis can be subclassified in three distinct clinical manifestations: aspergilloma, Aspergillus bronchitis/bronchiolitis and allergic infection. Insight is lacking why some PwCF do not develop Aspergillus disease upon exposure, while others develop hyper-inflammatory disease characterised by a Th1-driven inflammation or Th2-driven allergic hyper-inflammation. Over time, PwCF can present with different Aspergillus disease presentations and move between one entity and the other, which is an observation not yet explained.
Pulmonary aspergilloma
An aspergilloma is the development of a fungal ball in a pre-existing cavitary lesion or ectatic bronchus without signs of tissue invasion [70, 71]. Complex aspergilloma is nowadays referred to as chronic cavitary pulmonary aspergillosis, indicating multiple pulmonary cavities containing one or more aspergillomas [72, 73]. In PwCF, this is a rare complication that can present in those patients colonised with A. fumigatus and severe or end-stage pulmonary disease [74, 75], and in patients with large cystic bronchiectasis. Aspergilloma can be discovered on computed tomography (CT) as an accidental finding or after an episode of haemoptysis without any specific other symptoms [37, 76].
Aspergillus bronchitis/bronchiolitis
Invasive pulmonary aspergillosis (IPA) is defined as a condition where the hyphae invade the bronchial wall or adjacent lung parenchyma, such as the pulmonary vasculature [77, 78]. IPA is only seen in the case of immunodeficiency and as a rare entity in CF patients. In PwCF, Aspergillus bronchitis/bronchiolitis is the more common infection type. Aspergillus bronchitis, first reported in 2006 in a cohort of six CF patients [79], is defined as a chronic superficial infection of the airways [80]. The reported prevalence of Aspergillus bronchitis is 9% in PwCF, but likely to be under-reported [81]. Aspergillus bronchitis is defined as: a) symptoms related to chronic lower airway disease; b) Aspergillus species detected in sputum or BAL by culture or real-time polymerase chain reaction test; and c) Aspergillus-specific IgG in serum [80]. Clinical symptoms associated with Aspergillus bronchitis are productive cough, tenacious sputum, breathlessness and haemoptysis and can hardly be distinguished from a bacterial exacerbation in PwCF [40, 80].
Allergic pulmonary aspergillosis infection (ABPA)
ABPA is a serious complication in PwCF and the result of a complex immunologic response against A. fumigatus. ABPA has a prevalence of 4–9% in children and around 10% in adult PwCF [11, 82–88]. ABPA individuals sensitised to A. fumigatus can develop an IgE-mediated allergic inflammatory reaction (type I immediate hypersensitivity) which leads to an acute inflammatory response resulting in mucus impaction and bronchial obstruction after inhaling A. fumigatus spores [89–91]. ABPA mice models show that chronic exposure to A. fumigatus antigens may lead to fibrosis and irreversible lung damage [10, 89].
The diagnosis of ABPA can be difficult in PwCF because of atypical clinical symptoms such as cough, wheeze and increased sputum production [44, 92, 93]. Patients typically show a drop in forced expiratory volume in the first second that does not respond to regular antibiotic treatment directed against bacteria cultured from their sputum. The diagnosis can be supported by typical immunologic features such as an elevated total IgE concentration (greater than 1000 ng·mL−1 or 416 IU·mL−1), increased Aspergillus specific IgE antibodies (greater than 1.91 kilo units of antibody per litre), or increased Aspergillus specific IgG antibodies (greater than 78 mg·L−1) [25, 37, 40, 89, 94–100]. The basophil activation test, an in vitro test to measure the activation of basophils by IgE stimulation with A. fumigatus extract and CD63, CD193 and CD203c [101], can be used to discriminate ABPA from Aspergillus colonisation and non-ABPA in PwCF [102]. Furthermore, the results of basophil activation tests are not affected by antifungal or corticosteroid treatment; therefore, this test might be used as an additional criterion to identify ABPA and monitor A. fumigatus-related clinical status in PwCF [102–104].
Imaging and aspergillosis
Despite the currently available diagnostic tools, it remains challenging to differentiate colonisation versus infection with Aspergillus according to clinical tests (table 1). In clinical practice, it is also difficult to distinguish progression of CF lung disease due to Aspergillus from progression related to other, mainly bacterial, airway pathogens. Therefore, an important goal for clinicians is to find the particular fingerprint for Aspergillus-related lung damage (figure 2).
Imaging is an essential tool in the management of patients with pulmonary disease related to A. fumigatus [105, 106]. The two most used imaging tools to screen patients at high risk for pulmonary aspergillosis are chest radiography (CR) and CT. Both techniques are used to detect pulmonary disease related to A. fumigatus in patients with compatible illnesses, differentiate aspergillosis from other diseases, guide interventional procedures aimed at establishing a specific diagnosis and assess treatment response. CT is much more sensitive and specific than CR [107] in the evaluation of chest lesions and therefore considered the gold standard for an accurate differential diagnosis of different forms of pulmonary aspergillosis. CR, however, because of its high accessibility, low cost and radiation exposure to the patient, is frequently used when high frequency repetitive or bedside imaging of critically ill patients is required. More recently, magnetic resonance imaging (MRI) has been proposed as an imaging tool to diagnose specific subtypes of pulmonary aspergillosis [108–111]. A main advantage of MRI over CT is the ability to follow-up response to treatment with serial imaging without concerns related to radiation exposure. MRI has been increasingly used in those tertiary centres with experience in chest MRI.
In the section below we will discuss the most common radiological hallmarks of A. fumigatus colonisation, Aspergillus bronchitis and ABPA using CT and MRI in PwCF. Imaging features of each subtype of Aspergillus infection in PwCF are summarised in table 1.
Imaging features of A. fumigatus colonisation
In theory, one would expect that mere colonisation with A. fumigatus would not be related to any identifiable abnormalities on CT. In contrast, the few imaging studies that have looked into the relationship between A. fumigatus colonisation and structural lung changes in CF did report significant differences [2, 58]. In particular, PwCF who are colonised with A. fumigatus were found to have more low attenuation regions (often referred to as “trapped air”) on expiratory chest CT than a control group without colonisation [58]. There are also indications that this process can start early in life. In a study of a 5-year-old PwCF, the presence of A. fumigatus in the BAL fluid was associated with low attenuation regions on chest CT [58]. In addition, more severe bronchiectasis and more mucus plugging were observed in a group of A. fumigatus colonised PwCF compared to a non-colonised group [2]. Causal associations between A. fumigatus and the reported structural lung abnormalities cannot be asserted from these observational studies, but they do strongly suggest that airway cultures positive for A. fumigatus may not be an innocent finding in asymptomatic patients. Hence, at least a proportion of patients who are considered to be merely colonised by A. fumigatus according to current standards might in fact have an A. fumigatus infection.
Imaging features of saprophytic Aspergillus infection
Saprophytic aspergillosis or aspergilloma can be identified on chest CT as a fungal ball in a pre-existing pulmonary cavity, cyst or other air-containing space [97, 112]. Single or multiply lesions of aspergilloma can affect both lungs with equal frequency [113]. The mean diameter of aspergillomas is around 4 to 5 cm, with a maximum size of around 10 cm, and rarely shows regression in size without treatment [113].
An early CT finding of aspergilloma in the non-CF population and PwCF is the thickening of the lateral cavity wall, which reflects the saprophytic germination of the fungus onto the cavity wall. When this germination is large enough it will fall from the wall and form the aspergilloma (figure 3). The position of the fungal ball in the cavity on CT may change depending on the posture of the subject [78]. Pleural thickening can also be found on CT in association with A. fumigatus infection, which may present as early as 2–3 years before the formation of the fungal ball [78, 114, 115]. On MRI, the characteristic of aspergilloma shows low signal intensity on T1- and variable signal on T2-weighted images caused by calcium, air or ferromagnetic elements in the aspergilloma [116]. Air surrounding a fungal ball that does not completely fill up the cavity can be recognised as the Monod sign (figure 3) [78].
Imaging features of Aspergillus bronchitis/bronchiolitis
Histology of patients diagnosed as having Aspergillus bronchitis show superficial invasion by A. fumigatus of the superficial mucosal layers [21]. Therefore, CT findings in Aspergillus bronchitis follow the bronchocentric invasion of the airways. The initial signs of infection are bronchial wall thickening and centric-lobular nodules with ground glass opacities (GGOs) (figure 4a) with development of peri-bronchiolar and lobar consolidations as the infection progresses with consequent filling of airspaces (figure 4b). However, these findings can be non-specific, especially in PwCF [77, 78, 117]. The same holds for bronchiectasis, which has been described on CT in Aspergillus bronchitis in patients with non-CF chronic lung disorders, but is non-specific in PwCF [117]. To our knowledge there is only one case report on Aspergillus bronchitis in CF with a detailed description of CT findings, reporting signs of bronchitis/bronchiolitis with centrilobular nodules, tree-in-bud, GGO and peribronchial consolidations [118].
Allergic pulmonary aspergillosis
The most common radiological sign in ABPA is central varicose or cystic bronchiectasis in both non-CF patients and PwCF [10, 84, 91, 98, 119], also described as a “string of pearls” [120]. Another imaging sign of ABPA is mucous impaction in the central airways [99]. Furthermore, high attenuation regions on CT, with a mucus density higher than the para-spinal skeletal muscle (generally >70 Hounsfield units), are considered a specific feature for the radiological diagnosis of ABPA in CF [41, 84, 86, 121–125] (figure 5a, b). Nodule or granuloma formation can be detected in the periphery of the lungs [85], as well as large opacities, which tend to affect the upper and middle lobes [90, 126]. In addition, improvement of the imaging abnormalities in response to steroid treatment supports the diagnosis of ABPA in PwCF [89, 126].
General features of CF lung disease and early changes related to ABPA are difficult to differentiate. For the diagnosis and follow-up of ABPA, MRI has advantages over CT as it does not require ionising radiation. Furthermore, MRI allows the collection of morphological and functional information that might allow a more sensitive and specific diagnosis of ABPA-related changes [116]. Some studies with a small sample size have reported MRI findings of ABPA, especially in paediatric patients [108, 109]. These studies indicate that high T1 and low T2 signal intensities of mucus impaction (inverted impaction mucus sign) are both sensitive and specific for the diagnosis of ABPA in CF (figure 5c, d) [108–111]. This high discriminating ability of ABPA by MRI requires further confirmation in a larger number of PwCF.
Without antifungal treatment, Aspergillus diseases may move to chronic infections with complex appearances on image exams. The granulomas presenting in chronic Aspergillus infection indicate limited tissue invasion, which is often accompanied by enlarged intra-pulmonary lymph nodes (figure 6). On CT imaging, Aspergillus granulomas may cause extensive parenchymal consolidations, may lead to bronchiectatic cavities or may remain exclusively bronchogenic. The latter pattern is characterised by multiple nodules with a perilymphatic distribution (subpleural and perivisceral). Adjacent pleura can be invaded as well, which leads to the formation of cavitations, aspergilloma and pleural thickening on CT [73].
The way forward
Based on what is known in the literature, there are strong indications that A. fumigatus can contribute to the progression of structural lung damage in PwCF. As A. fumigatus can be cultured in a high percentage of PwCF, it is important to differentiate colonisation versus infection and to develop state-of-the-art sensitive and specific image analysis systems that are able to identify A. fumigatus-associated structural abnormalities on chest CT and/or MRI. This will support differentiation between harmless colonisation and infection with its associated structural lung damage. Such an image analysis system can be used for PwCF and A. fumigatus-positive sputum cultures to assist in the early detection and monitoring of A. fumigatus-associated structural lung damage and to monitor the effect of antifungal treatment.
Routine biennial chest CTs are used in around half of the European CF Society (ECFS) clinical trial network (CTN) centres. All these ECFS-CTN centres contribute data to the ECFS patient registry. The availability of an automated image analysis system sensitive and specific to detect A. fumigatus-associated structural changes opens up the possibility to analyse a large number of chest CTs to add key imaging outcomes to the registry. This will allow a better understanding of A. fumigatus induced lung damage. In addition, such a system can play an important role as outcome measures for clinical studies. An example of this is the recently started international cASPerCF study (clinicaltrial.gov identifier NTC 01782131) [127] evaluating the therapeutic effect of posaconazole treatment in children and adolescents with CF and A. fumigatus infection. The cASPerCF study will explore whether chest CT-related outcomes are sensitive to detect a therapeutic effect.
Furthermore, chest MRI could also play an important role in the differential diagnosis and follow-up of pulmonary aspergillosis. Chest MRI could be used as an imaging tool for short-term follow-up of therapy changes in PwCF. In our centre, we are currently using a short scanning protocol including T2-weighted and proton density-weighted imaging to follow treatment changes in PwCF and Aspergillus infection, namely consolidation and large mucus plugs. The complementary role of chest MRI in pulmonary aspergillosis will become clinically relevant when this technique has broader use in clinical practice. Current limitations to the use of MRI in clinical practice are the lack of protocol standardisation between CF centres and major differences in image quality, which hampers its applicability on a large scale. Finally, post-processing tools for chest MRI are not commercially available and most of the time are reported in the literature as in-house developed tools.
Conclusion
Our review provides support that specific structural lung damage on chest CT associated with A. fumigatus infection in PwCF can be identified. The development of sensitive and specific image analysis methods for chest CT and MRI are needed to identify whether in PwCF A. fumigatus-related structural lung damage can be detected at an early stage to guide treatment. Furthermore, image-related outcomes can be important as outcome measures for clinical studies and for clinical management.
Acknowledgements
We would like to acknowledge W.M. Bramer (Erasmus MC medical library) for his assistance in the literature search. We would like to thank T. van der Veer (Erasmus MC) for providing figure 3.
Footnotes
Provenance: Submitted article, peer reviewed.
Author contributions: Q. Lv contributed to the design of the work, drafting the manuscript, drawing the figure, and gave final approval to the manuscript. B.B.L.J. Elders contributed to critically revising the manuscript and gave final approval to the manuscript. A. Warris contributed to critically revising and gave final approval to the manuscript. D. Caudri contributed to revising the manuscript, drawing the figure, and gave final approval to the manuscript. P. Ciet contributed to the design of the work, picture selections and gave final approval to the manuscript. H. Tiddens contributed to the design of the work, critically revised the manuscript, and gave final approval to the manuscript.
Conflict of interest: Q. Lv has nothing to disclose.
Conflict of interest: B.B.L.J. Elders has nothing to disclose.
Conflict of interest: A. Warris has nothing to disclose.
Conflict of interest: D. Caudri has nothing to disclose.
Conflict of interest: P. Ciet reports personal fees from Vertex Pharmaceutical, outside the submitted work.
Conflict of interest: H. Tiddens reports grants and other funding from Novartis, grants from CFF and Vectura, and personal fees from Vertex, Thirona and Insmed, outside the submitted work. In addition, Erasmus MC and Telethon Kids Institute have licensed the use of PRAGMA-CF to Thirona and Resonance Health. The Sophia research BV of the Erasmus MC-Sophia Childrens hospital has received unconditional research grants from Novartis, and Vectura.
Support statement: Funding was received from Nederlandse Cystic Fibrosis Stichting (NCFS)–Health Holland (PPS). Funding information for this article has been deposited with the Crossref Funder Registry.
- Received April 23, 2021.
- Accepted August 7, 2021.
- Copyright ©The authors 2021
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