Diagnosis of invasive aspergillosis

INTRODUCTION — The term "aspergillosis" refers to illness due to allergy, airway or lung invasion, cutaneous infection, or extrapulmonary dissemination caused by species complexes of Aspergillus, most commonly A. fumigatus, A. flavus, A. niger, and A. terreus. Aspergillus species are ubiquitous in nature, and inhalation of infectious conidia is a common event. However, tissue invasion is uncommon and occurs most frequently in the setting of immunosuppression associated with receipt of therapy for hematologic malignancies or hematopoietic cell or solid organ transplantation.

The diagnosis of invasive aspergillosis will be reviewed here. The epidemiology, clinical manifestations, and treatment of invasive aspergillosis, as well as the diagnosis of other syndromes caused by Aspergillus spp, are presented elsewhere. (See "Epidemiology and clinical manifestations of invasive aspergillosis" and "Treatment and prevention of invasive aspergillosis" and "Clinical manifestations and diagnosis of allergic bronchopulmonary aspergillosis" and "Chronic pulmonary aspergillosis: Epidemiology, clinical manifestations and diagnosis".)

APPROACH TO DIAGNOSIS

General approach — Culture of Aspergillus spp in combination with the histopathologic demonstration of tissue invasion by hyphae provides definitive evidence of invasive aspergillosis [1,2]. However, biopsy is frequently not feasible due to the risks of complications (eg, bleeding risk in patients with thrombocytopenia).

A rational first step to establishing the diagnosis of invasive aspergillosis involves the use of less invasive modalities, such as serum biomarkers (galactomannan, beta-D-glucan, and polymerase chain reaction [PCR] assays), and obtaining sputum and/or bronchoalveolar lavage (BAL) specimens for fungal staining, fungal culture, BAL galactomannan, and/or PCR.

A positive sputum fungal stain and/or culture should prompt therapy of hosts who are at risk for invasive aspergillosis. In the right clinical context, a positive BAL galactomannan antigen, BAL PCR for aspergillus species, or a positive serum galactomannan antigen can also provide a presumptive diagnosis [2-4]. In contrast, a positive serum beta-D-glucan assay can occur in the setting of various invasive fungal infections, including candidiasis.

Patients with clinical and radiographic findings that are suggestive of an invasive fungal infection, but in whom noninvasive testing and/or bronchoscopy is unrevealing, should ideally undergo lung biopsy.

Options for biopsy include bronchoscopy with transbronchial biopsy, computed tomography-guided transthoracic needle biopsy, and video-assisted thorascopic surgery. The most appropriate technique depends upon the location of the lesion(s), the individual patient's risk of complications from each procedure, and the necessity to establish the diagnosis. The decision regarding the need for a histopathologic diagnosis must be made on a patient-by-patient basis.

Approach in patients with COVID-19 — An increased incidence of pulmonary aspergillosis has been reported in patients with coronavirus disease 2019 (COVID-19) infection, most frequently in patients with severe COVID-19 who are mechanically ventilated, are >65 years of age, have underlying lung disease, and/or have immunocompromising conditions [5,6]. (See "Epidemiology and clinical manifestations of invasive aspergillosis".)

The diagnosis of pulmonary aspergillosis in patients with COVID-19 is challenging because they may lack the classic risk factors for invasive aspergillosis (ie, hematologic malignancies, neutropenia) and radiographic findings [7-9].

Suspicion for invasive aspergillosis should be raised in any patient with COVID-19 who has radiographic findings that suggest aspergillosis (ie, nodular opacities with surrounding ground glass infiltrates or cavitary disease). Because typical radiographic findings may be absent or obscured by those due to COVID-19, suspicion should also be raised in any patient with COVID-19 who has prolonged or relapsing respiratory failure without alternate explanation.

When invasive aspergillosis is suspected, we typically perform a bronchoscopy to directly visualize the airways and perform BAL to obtain samples for microbiologic diagnosis. In general, we perform fungal staining, fungal culture, galactomannan antigen, and/or PCR for Aspergillus on the BAL fluid. While the diagnostic utility of serum biomarkers is less clear in patients with COVID-19, we usually obtain both a serum galactomannan test and a beta-D-glucan test because positive results can help corroborate the diagnosis of invasive aspergillosis. Serum PCR testing should be performed, if available. These tests are typically performed as part of a more comprehensive evaluation for respiratory failure. A blind nonbronchoscopic lavage can be obtained if bronchoscopy is not feasible due to infection control concerns or other factors [9]. Tracheal aspirates can be used in conjunction with other assessments, but positive culture results and/or galactomannan results may reflect colonization rather than invasive disease [7,9]. (See "Clinical presentation and diagnostic evaluation of ventilator-associated pneumonia", section on 'Invasive respiratory sampling'.)

For patients who have radiographic findings consistent with aspergillosis (ie, nodules or cavities) and lack of concern for other causes, detection of aspergillosis by culture, BAL or serum galactomannan, and/or PCR is typically sufficient for diagnosis. When consistent radiographic findings are absent, it is difficult to determine whether the detection of Aspergillus in the airways represents colonization or active infection; in such cases, we typically make the decision to treat on a case-by-case basis.

Our approach is similar to that outlined by an expert task force on the diagnosis and clinical management of COVID-19 associated pulmonary aspergillosis [8,9].

Treatment is discussed separately. (See "Treatment and prevention of invasive aspergillosis".)

DIAGNOSTIC MODALITIES — Aspergillus species are frequently inhaled into the airways, but because of effective conidial clearance in the majority of individuals, disease usually does not result. Because we inhale conidia constantly, culture isolation of Aspergillus species from the airway does not necessarily indicate disease. Thus, the diagnosis of invasive aspergillosis is based upon both isolating the organism (or markers of the organism) and the probability that it is the cause of disease. The latter is a function of the host's risk factors for disease (eg, immune status) and the clinical presentation. Demonstration of hyphal elements invading tissues from biopsy of any affected site, such as the lung or skin, represents a proven diagnosis [10].

Given the above issues, the diagnosis of invasive aspergillosis is often referred to within a scale of certainty: possible, probable, or proven [2,10,11]. These definitions have been developed in order to maintain consistency in clinical and epidemiologic studies, not to drive therapeutic decision making.

Direct examination of respiratory specimens — Respiratory specimens are usually stained with calcofluor white with 10% potassium hydroxide to detect the presence of fungal elements [12]. Gomori methenamine silver can be used to stain cytology preparations. Organisms can be observed as narrow (3 to 6 microns wide), septated hyaline hyphae with dichotomous acute angle (45°) branching [13]. However, several filamentous fungi, including Scedosporium spp and Fusarium spp, have similar appearances to Aspergillus spp on direct microscopy.

Culture — Culture of the organism, in combination with evidence of tissue invasion on histopathology or culture from a normally sterile site, provides the most certain evidence of invasive aspergillosis [10]. However, both microscopic examination and culture are insensitive [14], and therapy should not be withheld in the absence of such confirmation. Furthermore, performing a biopsy is not feasible in some patients due to bleeding risk or risk of other complications. In patients with risk factors and clinical and/or radiographic features that are suggestive of invasive aspergillosis, culture of Aspergillus spp from respiratory secretions provides adequate evidence of invasive disease.

Aspergillus is a rapidly growing fungus in the laboratory and is often visible in culture within one to three days of incubation. Sporulation is required for examination of spore-bearing structures, and diagnosis at the species complex level. Molecular testing is required for definitive diagnosis of the specific species within the complex.

Occasionally, organisms can be difficult to identify due to slow sporulation. Slow sporulating organisms may have been disregarded as non-pathogens in the past. However, more recently, slow-sporulating species (eg, Aspergillus lentulus, Neosartorya udagawae, and others) with variable susceptibility profiles have been identified and implicated in invasive infections [15]. These organisms should not be disregarded as contaminants based on the slow-sporulating phenotype. (See "Treatment and prevention of invasive aspergillosis", section on 'Consideration of antifungal resistance'.)

Many patients with documented invasive aspergillosis have negative cultures. This has been observed in surveillance studies, and the prevalence of negative cultures varies depending upon the population being evaluated. As an example, in multicenter surveillance studies, only 25 to 50 percent of hematopoietic cell transplant (HCT) recipients who met criteria for invasive aspergillosis based upon galactomannan antigen results had positive cultures [16,17].

The predictive value of positive sputum or bronchoalveolar lavage (BAL) cultures is dependent upon both the host and the clinical presentation. In a study that evaluated the predictive value of lower respiratory tract cultures in different patient populations with probable or proven invasive pulmonary aspergillosis, the positive predictive value was highest in HCT recipients, patients with hematological malignancies, and granulocytopenic patients (72 percent), compared with solid organ transplant recipients and patients receiving glucocorticoids (58 percent) and patients with HIV (14 percent) [18]. The positive predictive value was highest in BAL cultures, in part because the prevalence of invasive aspergillosis is higher among patients who have radiographic abnormalities warranting bronchoscopy. Clinical and radiographic findings suggestive of invasive aspergillosis were present significantly more often among infected patients compared with uninfected patients.

Histopathology — In the setting of invasive disease, organisms can be observed in biopsy specimens as narrow (3 to 6 microns wide), septated hyaline hyphae with dichotomous acute angle (45°) branching (picture 1 and picture 2 and picture 3). The organism can be seen using Gomori methenamine silver or periodic acid-Schiff staining. However, several hyaline molds, including Scedosporium spp and Fusarium spp, have similar appearances to Aspergillus spp in histopathologic sections. Since the treatment of infections caused by these fungi may differ, it is important to confirm genus and species by culture. (See "Clinical manifestations and diagnosis of Fusarium infection", section on 'Diagnosis' and "Epidemiology, clinical manifestations, and diagnosis of Scedosporium and Lomentospora infections", section on 'Histopathology'.)

Histopathologic examination can sometimes distinguish the above organisms from the order Mucorales(the agents of mucormycosis), which appear as broad, nonseptate hyphae that exhibit right-angle branching (picture 4 and picture 5). Occasionally, this distinction can be difficult, especially when small hyphal fragments are present or when the organism folds back on itself to create "pseudo-septations." Determining whether the fungus is one of the Mucorales or another fungus is important because the Mucorales are not susceptible to voriconazole, which is the treatment of choice for invasive aspergillosis. (See "Mucormycosis (zygomycosis)" and "Treatment and prevention of invasive aspergillosis".)

Galactomannan antigen detection — Galactomannan is a polysaccharide that is a major constituent of Aspergillus cell walls. A double sandwich enzyme immunoassay (EIA) that utilizes the monoclonal antibody EB-A2 forms the current commercial (Platelia) assay, which has been approved by the US Food and Drug Administration (FDA) for testing serum and BAL fluid. This antibody detects multiple epitopes on galactofuranose side chains that are linked to the large polymer mannan backbone [19]. Galactofuranose is the six-member ring form of galactose, which is made by non-mammalian eukaryotes and some prokaryotic pathogens, and is a common antigenic moiety of different glycoconjugates [20]. Although once described as being Aspergillus specific, we now know that galactofuranose is present in other fungi and certain other substances. (See 'Caveats' below.)

Interpretation — The galactomannan EIA is performed with an optical read-out that is interpreted as a ratio relative to the optical density (OD) of a threshold control provided by the manufacturer; this ratio is called the OD index. Initial use of the test, largely in Europe, utilized a relatively high OD index (1 to 1.5) as a cut-off for positivity in order to ensure few false-positive results. Subsequent studies demonstrated that lower thresholds (0.5 to 0.7) provide relatively better performance [21,22]. The assay cleared by the FDA has a suggested threshold OD index of 0.5; thus, an OD index ≥0.5 is considered to be a positive result. For clinical trial purposes, in order to ensure a higher likelihood of diagnostic certainty, a threshold OD index of ≥1.0 in serum or plasma has been proposed and included in the European Organisation for Research and Treatment of Cancer (EORTC)/Mycoses Study Group Education and Research Consortium (MSGERC) definitions [2,23].

Serum — In some patients, galactomannan antigen can be detected in the serum before the presence of clinical signs or symptoms of invasive aspergillosis. Several studies have assessed its test characteristics, and the performance of the test has been addressed in a review and meta-analysis [24,25]. Some inconsistency in the reported performance of the test has been introduced by studies that used different cut-offs for positivity as well as other variables, such as different populations evaluated and testing on patients receiving concurrent antifungal drugs. One review noted that the sensitivity of the test varied from 30 to 100 percent, whereas specificity remained relatively high and constant (>75 percent) [26].

A meta-analysis that included 50 studies with a total of 5660 immunocompromised patients, including 586 with proven or probable invasive aspergillosis, reported that using an OD index cutoff of 0.5, sensitivity of the galactomannan EIA was 82 percent (95% CI 73-90 percent) and specificity was 81 percent (95% CI 72-90 percent) [25].

In another meta-analysis, subgroup analyses suggested that the assay performs better in patients who have a hematologic malignancy or who have received HCT; in comparison, the test performance may be limited in solid organ transplant recipients [27]. Whether this is related to biologic differences in the type of invasive disease, such as degree of fungal burden, is unclear. Few cases were included in the studies that have been performed; more patients who have different underlying diseases will need to be studied to establish the usefulness of this assay in disparate populations.

Caveats — The following caveats should be considered when using the galactomannan EIA:

●The sensitivity of detecting galactomannan in serum is decreased by concurrent administration of mold-active antifungal therapy [28-30].

●In the past, false-positive serum results were demonstrated in patients who were receiving intravenous piperacillin-tazobactam due to the presence of galactomannan (or a cross-reactive antigen) in the antibiotic formulation [31,32]. False-positive results persisted for as long as five days after discontinuation of piperacillin-tazobactam [32,33]. Results of more recent studies suggest that current preparations of piperacillin-tazobactam rarely react with the assay [34,35]. False-positive galactomannan results have also been reported with intravenous amoxicillin-clavulanate, a formulation that is not available in the United States [36].

●Many fungi demonstrate galactomannans (or polysaccharides containing galactofuranose residues) on their cell walls. False-positive results of the Aspergillus galactomannan EIA may be seen with infections caused by organisms that share cross-reacting antigens. These include filamentous Ascomycetes that cause similar disease presentations (eg, Fusarium species [37]) and others (Penicillium species [38], Histoplasma capsulatum [39]).

●False-positive results are more likely to occur during the first 100 days following HCT and in patients with gastrointestinal tract mucositis caused by chemotherapy or graft-versus-host disease (GVHD) [40]. The proposed mechanism is that galactomannan in foods or bacteria having cross-reactive epitopes may translocate across the intestinal mucosa during periods of impaired mucosal integrity [31].

●Another possible cause of false-positive galactomannan EIA results is contamination of foods with Aspergillus or closely related fungi, such as Penicillium spp. One report noted false-positive results associated with ingestion of large numbers of frozen ice-pops in the setting of gastrointestinal GVHD [41]. Possible causes included Penicillium contamination of the ice-pop wrappers or a food additive in the ice pops, such as sodium gluconate.

●False-positive results have been reported in patients who received transfusions of blood products that were collected in bags produced by a single manufacturer (Fresenius Kabi, Germany) but not in patients who received blood products that were collected in bags produced by other manufacturers [42].

●In one study, some patients who received intravenous immunoglobulin had a false-positive galactomannan EIA result [43].

●Some false-positive results have been reported in children, in whom the assay has been less well studied. However, other studies suggest relatively little false positivity, with similar results to those in adults [44].

Role of testing — Studies evaluating the serum galactomannan EIA report relatively low (<50 percent) and high (>90 percent) positive and negative predictive values, respectively. These values are largely a function of the low prevalence of disease, even when measured in high-risk populations (usually <20 percent). One should thus consider the clinical context to determine the probability of infection [26]. (See "Glossary of common biostatistical and epidemiological terms", section on 'Predictive values'.)

Some investigators have suggested using the serum galactomannan EIA for screening (weekly or twice weekly) in order to detect invasive aspergillosis prior to the development of clinical signs or symptoms. Biomarker screening as a means to guide pre-emptive therapy was evaluated in a randomized trial; screening with galactomannan antigen and PCR led to reduced empiric antifungal treatment compared with a culture-based strategy [45]. The use of biomarker-driven strategies allows less empiric use of antifungal therapy for fever and more pre-emptive therapy based on positive biomarker results [1,2]. (See "Treatment and prevention of invasive aspergillosis", section on 'Pre-emptive therapy'.)

The utility of the serum galactomannan EIA has been best established in the setting of suspected disease in high-risk patients with hematologic malignancies. In the setting of clinical disease suspicion, prevalence is higher, assuring better predictive performance. The assay may also be useful in other immunocompromised patients at risk for invasive pulmonary aspergillosis, but caution is necessary in interpreting results as sensitivity is not as high in patients who have disease that may be limited to the airways (eg, lung transplant recipients). (See "Fungal infections following lung transplantation", section on 'Galactomannan, beta-D-glucan, and PCR'.)

Some investigators have suggested a role for serial galactomannan testing in patients with documented invasive aspergillosis, both as a prognostic measure of effectiveness of antifungal therapy and to differentiate between worsening fungal infection and worsening radiographic findings developing as a result of the host immune response [46-50]. The prognostic value of the serum galactomannan EIA is discussed separately. (See "Treatment and prevention of invasive aspergillosis", section on 'Prognostic factors'.)

Bronchoalveolar lavage fluid — The galactomannan EIA detects fungal antigens even when the organism does not grow in the laboratory, providing an indication of potentially invasive disease. The galactomannan EIA performed on BAL fluid provides additional sensitivity compared with culture, estimated in most studies to exceed 70 percent [51-55].

The optimal threshold OD index for positivity continues to be debated; a higher threshold OD index results in lower sensitivity but higher specificity for invasive aspergillosis. In a meta-analysis of studies of BAL galactomannan in immunocompromised patients, an OD cutoff of 0.5 resulted in a sensitivity of 0.88 and a specificity of 0.81, whereas a cutoff of 1.0 resulted in a sensitivity of 0.78 and a specificity of 0.93 [55]. As noted above, the FDA considers an OD index of ≥0.5 to be positive for galactomannan EIA in both serum and BAL fluid, although a revised threshold of 1.0 for BAL fluid is now included in the EORTC/MSGERC definitions [2,23]. Higher OD levels are associated with greater diagnostic certainty.

It is important to note that false-positive results occur, and these are especially common in the setting in which the detection of fungi in the airways represents colonization as occurs in lung transplant recipients or when the fluid that is used for BAL washes is contaminated with galactomannan. However, one study performed in lung transplant recipients suggested that specificity is high (95 percent) [51]. Many centers have begun to utilize this assay on BAL fluid as an adjunct to other diagnostic tests when invasive aspergillosis is suspected. Because performance may be dependent on technical variables, such as type and amount of BAL fluid, institutions should establish specific protocols for testing.

The use of the galactomannan assay on BAL fluid from lung transplant recipients is discussed in greater detail separately. (See "Fungal infections following lung transplantation", section on 'Galactomannan, beta-D-glucan, and PCR'.)

Other specimen types — Although the galactomannan assay has been approved by the FDA for use only on serum and bronchoalveolar lavage fluid, galactomannan can also be detected in other samples, including cerebrospinal fluid and pleural fluid, depending on the clinical context [56].

Beta-D-glucan assay — 1,3-Beta-D-glucan, a cell wall component of many fungi, is detected by the beta-D-glucan assay. There are several different commercial assays available in different countries. The Fungitell assay has been cleared by the FDA as an aid to diagnose invasive fungal infections. These assays utilize the same principle as serum endotoxin assays, measuring activation of Factor G. Since the different marketed tests utilize different horseshoe crab substrates and different methods to measure output, performance may be variable. The output of the serum assay currently available in the United States is based on spectrophotometer readings, in which optical density is converted to beta-D-glucan concentrations; the results are interpreted as negative (range <60 pg/mL), indeterminate (60 to 79 pg/mL), or positive (>80 pg/mL) [57]. Importantly, these cut-offs were defined in the clinical context of identifying breakthrough invasive fungal infections (primarily, invasive candidiasis) in people who were undergoing treatment for hematologic malignancies. Precise cut-offs to optimize performance of the assay as an aid to diagnose invasive aspergillosis have not been defined and may be different. In a 2011 meta-analysis that included 16 studies evaluating beta-D-glucan assays for the diagnosis of invasive fungal infections, the pooled sensitivity was 77 percent (95% CI 67-84 percent) and the pooled specificity was 85 percent (95% CI 80-90 percent) [58]. A 2012 meta-analysis that included six cohort studies of patients with hematologic malignancies noted a lower sensitivity (50 percent, 95% CI 34-65 percent) and a higher specificity (99 percent, 95% CI 97-100 percent) than previously reported; however, there were few cases of invasive aspergillosis tested to provide definitive conclusions [59]. In individual studies, the sensitivity has ranged from 55 to 95 percent and the specificity has ranged from 77 to 96 percent [57,60-64]. As in studies evaluating other fungal diagnostics, likely reasons for the differences in sensitivity and specificity between studies are that different thresholds were considered positive, different assays were used, patient populations varied, and study design was not uniform. Despite the substantial heterogeneity among different studies, the beta-D-glucan assay has good accuracy for distinguishing patients with proven or probable invasive fungal infections from patients without invasive fungal infection [58].

One study compared performance of the galactomannan EIA with the beta-D-glucan assay in sera from 105 patients with invasive aspergillosis and 50 healthy blood donors [65]. Results demonstrated a higher specificity with the galactomannan test (97 versus 82 percent) but a lower sensitivity (81 versus 49 percent).

Caveats — The following caveats should be considered when using the beta-D-glucan assay:

●The beta-D-glucan assay is not specific for Aspergillus species and can be positive in patients with a variety of invasive fungal infections, including candidiasis and Pneumocystis jirovecii (formerly P. carinii); the latter can be associated with beta-D-glucan levels greater than the upper limits of the assay. Although the beta-D-glucan assay may be positive in patients with a variety of invasive fungal infections, it is typically negative in patients with mucormycosis or cryptococcosis [62,63,66,67]. (See "Epidemiology, clinical manifestations, and diagnosis of Pneumocystis pneumonia in patients without HIV", section on 'Beta-D-glucan assay'.)

●The specificity of the assay can be decreased by multiple other clinical variables; the following factors have been reported to cause false-positive results [66]:

•Hemodialysis with cellulose membranes

•Intravenous immunoglobulin

•Albumin

•Use of cellulose filters for intravenous administration

•Gauze packing of serosal surfaces [68]

•Intravenous amoxicillin-clavulanic acid (a formulation that is not available in the United States) [69]

•Infections with certain bacteria that contain cellular beta-glucans, such as Pseudomonas aeruginosa [70,71]

Role of testing — The beta-D-glucan assay may be used for detecting invasive fungal infections early in the course of infection, prior to the onset of overt clinical findings. One study evaluated a screening strategy in which 95 patients receiving chemotherapy for acute leukemia underwent beta-D-glucan testing twice weekly in the absence of fever and daily in the presence of fever [72]. Screening appeared to shorten the time interval between suspected infection and established diagnosis, when compared with waiting for clinical signs and symptoms of disease. However, this strategy has not been validated in randomized trials. Because of its limited sensitivity, beta-D-glucan testing should not be used to rule out invasive fungal infections, particularly in patients with hematologic malignancies due to the high prevalence of invasive fungal infections in this population [73]. (See "Treatment and prevention of invasive aspergillosis", section on 'Pre-emptive therapy'.)

Polymerase chain reaction — When invasive aspergillosis is suspected, we typically include PCR, in conjunction with other biomarkers, in our diagnostic evaluation. PCR can be performed on serum, plasma, whole blood, or BAL fluid. As with other biomarkers, diagnostic certainty rises with two consecutive positive results. In addition to diagnosis, some PCR-based assay can detect mutations associated with antifungal resistance [3].

Molecular assays (eg, by PCR) have undergone extensive evaluation [4,28,39,57,60,74-80]. In a meta-analysis of 25 studies, sensitivity and specificity of PCR to detect invasive aspergillosis were 84 and 76 percent, respectively [79]. When at least two PCR results were positive, the sensitivity was 64 percent and the specificity was 95 percent. Another meta-analysis had similar findings [80]. These results suggest that two positive PCR results are highly suggestive of invasive aspergillosis. Standardized protocols for nucleic acid extraction, sample types, volumes, and processing have been developed by the European Aspergillus PCR Initiative/Fungal PCR Initiative [3].

Assays under development — While multiple assays are under development, recent publications have focused on two tests as aids to diagnose aspergillosis:

●A lateral flow device (LFD) that detects a mannoprotein produced by actively growing Aspergillus species using a monoclonal JF5 antibody (AspLFD) has been found to have reasonable performance in patients at risk for aspergillosis (hematologic malignancy, solid organ transplant. In a case-control study, performance of the LFD compared with galactomannan and PCR was relatively comparable when applied to serum [81]. Other studies have compared performance of the LFD with the galactomannan and beta-D-glucan assays on BAL fluid, finding promising results in patients with hematologic malignancies, solid organ transplant, or respiratory disease [82,83]. The LFD assay is inexpensive, does not require special equipment, and can be performed quickly at the point of care [24]. An updated version of that assay was shown to improve clinical utility and is marketed as a diagnostic aid [84]. Another Aspergillus LFD detecting galactomannan in serum is Conformitè Europëenne cleared (sona Aspergillus galactomannan lateral flow assay) and shows good agreement with conventional galactomannan measurement [85]. Additionally, a lateral flow dipstick assay undergoing development which uses a galactofuranose-specific antibody successfully detected Aspergillus antigens in urine in preclinical studies, which could be a rapid and easy to perform point-of-use test [86].

●Another technology relies on detection of secondary metabolites in human breath using thermal desorption-gas chromatography/mass spectrometry. Results of a small prospective study that enrolled patients diagnosed with proven or probable invasive aspergillosis reported high sensitivity (94 percent; 95% CI 81-98 percent) and specificity (95% CI 79-98 percent) [87].

While these assays require more study to define performance parameters, they appear promising.

Combining assays — In a meta-analysis of studies that assessed the diagnostic performance of serum galactomannan and serum or whole-blood PCR as weekly screening in high-risk patients, single positive test results had good sensitivity (92 percent for galactomannan; 84 percent for PCR) and specificity (90 percent for galactomannan; 76 percent for PCR) for detecting proven or probable invasive aspergillosis [88]. However, when positivity was defined as at least one positive result (either galactomannan or PCR), the sensitivity was 99 percent. When both the galactomannan and the PCR results were positive for the same patient, the specificity was 98 percent; specificity was also high with two positive galactomannan results (95 percent) or two positive PCR results (93 percent). In an open-label randomized trial of high-risk patients with hematologic malignancies who were not receiving antifungal prophylaxis, treating physicians were informed of either serum galactomannan and PCR results or of serum galactomannan results only; testing was performed twice weekly [89]. Positivity of either assay prompted chest computed tomography (CT) scan and initiation of antifungal therapy. The use of the serum galactomannan assay and PCR for screening was associated with earlier diagnosis and a lower incidence of invasive aspergillosis than the use of the galactomannan assay alone.

Most studies comparing the performance parameters of different assays (the galactomannan EIA, beta-D-glucan assay, LFD, and PCR) for use on BAL fluid emphasize the insensitivity of culture for isolation of Aspergillus species and suggest that the best approach may be to combine assays [83]. In a total of 78 BAL fluid samples, the sensitivity of all four methods ranged from 70 to 88 percent. The combination of the galactomannan EIA and either PCR or LFD increased sensitivity to 94 to 100 percent without compromising specificity. The beta-D-glucan assay applied on BAL fluid resulted in high false-positive rates.

Precipitin antibodies — Detection of precipitin antibodies may be useful for the diagnosis of allergy to various molds, including Aspergillus, but has no role in the diagnosis of invasive aspergillosis. (See "Clinical manifestations and diagnosis of allergic bronchopulmonary aspergillosis", section on 'Diagnosis'.)

Imaging — Imaging is an important component of the diagnostic evaluation. The lungs are the most commonly affected site in invasive aspergillosis; CT scanning of the lungs often helps to support the diagnosis (image 1). In patients with clinical findings suggestive of sinus involvement, CT of the sinuses should be performed (image 2). When brain involvement is suspected, brain magnetic resonance imaging is indicated (image 3 and image 4).

The imaging findings associated with invasive pulmonary aspergillosis, such as nodules with surrounding hypoattenuation, termed the halo sign, can be seen with other angioinvasive pulmonary infections (image 1). However, this finding most often represents invasive aspergillosis because it is the most common mold to cause invasive infection. The "reversed halo" sign has been described to be potentially a more pathognomonic pattern for mucormycosis, as the inner zone of hypoattenuation corresponds with early progression of necrosis with the agents of mucormycosis [90]. Imaging findings in patients with invasive aspergillosis are presented in greater detail separately. (See "Epidemiology and clinical manifestations of invasive aspergillosis", section on 'Imaging'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Aspergillosis".)

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Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Basics topic (see "Patient education: Invasive aspergillosis (The Basics)")

SUMMARY AND RECOMMENDATIONS

●Colonization versus invasive disease – Aspergillus conidia are frequently inhaled into the airways but, because of effective clearance in the majority of individuals, disease usually does not result. Because we inhale conidia constantly, culture isolation of Aspergillus species from the airway does not necessarily indicate disease. Thus, the diagnosis of invasive aspergillosis is based upon both isolating the organism (or markers of the organism) and the probability that it is the cause of disease. (See 'Diagnostic modalities' above.)

●Diagnostic approach – A rational first step to establishing the diagnosis of invasive aspergillosis involves the use of noninvasive modalities, such as serum biomarkers (galactomannan, beta-D-glucan assays, and polymerase chain reaction [PCR] testing) and obtaining sputum for fungal staining and culture. If the diagnosis is not made by these methods, a more invasive approach is indicated when feasible. Options include bronchoscopy with bronchoalveolar lavage (BAL), transbronchial biopsy, computed tomography–guided transthoracic needle biopsy, and video-assisted thorascopic surgery. When BAL is performed, a sample should be sent for galactomannan antigen testing. (See 'Approach to diagnosis' above.)

Culture in combination with evidence of tissue invasion on histopathology or culture from a normally sterile site provides the most certain evidence of invasive aspergillosis. However, both microscopic examination and culture are insensitive, and therapy should not be withheld in the absence of such confirmation. (See 'Culture' above.)

●Biopsy findings – Aspergillus organisms observed in biopsy specimens are typically narrow (3 to 6 microns wide), septated hyaline hyphae with acute angle branching (picture 1 and picture 2). However, several hyaline molds including Scedosporium spp and Fusarium spp have similar appearances to Aspergillus spp in histopathologic sections. The treatment of infections caused by these fungi may differ, so it is important to obtain cultures to confirm genus and species. (See 'Histopathology' above.)

Histopathologic examination can usually distinguish Aspergillus spp and the other fungi described above from the Mucorales, which appear as broad, nonseptate hyphae that exhibit right angle branching (picture 4 and picture 5). Determining whether the fungus is a Mucorales is important because these molds are not susceptible to voriconazole, which is the treatment of choice for invasive aspergillosis. (See 'Histopathology' above.)

●Diagnosis when biopsy is not feasible – Performing a biopsy is not feasible in some patients due to the risk of bleeding or other complications. In patients with risk factors and clinical and/or radiographic features that are suggestive of invasive aspergillosis, culture of Aspergillus spp from respiratory secretions or finding typical hyphae on staining of respiratory secretions provides adequate evidence to warrant therapy. (See 'Culture' above.)

●Role of biomarkers

•Galactomannan – Galactomannan is a major constituent of Aspergillus cell walls. A double sandwich enzyme immunoassay that detects the galactomannan antigen by binding to galactofuranose side chains is available for use on serum and BAL fluid as an adjunctive test for the diagnosis of aspergillosis. The utility of the serum galactomannan assay has been best established in the setting of suspected disease in patients with hematologic malignancies. (See 'Galactomannan antigen detection' above.)

•Beta-D-glucan – 1,3-Beta-D-glucan, a cell wall component of many fungi, is detected by the beta-D-glucan assay. However, this assay is not specific for Aspergillus species and can be positive in patients with a variety of invasive fungal infections, including candidiasis and Pneumocystis jirovecii (formerly P. carinii). (See 'Beta-D-glucan assay' above.)

•Polymerase chain reaction (PCR) – PCR for Aspergillus DNA can be performed on serum, plasma, whole blood, and/or BAL fluid. As with other biomarkers, a positive result can provide a presumptive diagnosis in the right clinical context; diagnostic certainty rises with two or more consecutive positive results. (See 'Polymerase chain reaction' above.)

ACKNOWLEDGMENTS — The UpToDate editorial staff acknowledges Kieren A Marr, MD and Daniel J Sexton, MD, who contributed to an earlier version of this topic review.