Introduction
Obstructive pulmonary diseases (OPD) (asthma and chronic obstructive pulmonary disease (COPD)) are prevalent chronic respiratory system ailments characterized by progressive airflow limitation, pulmonary inflammation, and airway remodelling [1, 2]. Patients with OPD often exhibit pathological changes such as airway inflammation, increased mucus secretion, and diminished pulmonary immune function, rendering them susceptible to various respiratory pathogens [3]. Among them, invasive pulmonary aspergillosis (IPA) is a common complication of OPD, posing a severe threat to patients’ lives. Fungi of the Aspergillus genus, particularly Aspergillus fumigatus, are commonly found pathogens in OPD patients [4]. In individuals with OPD, due to airway inflammation and compromised immune resistance, Aspergillus fumigatus can easily invade lung tissues, triggering IPA. Clinical manifestations of IPA vary, including symptoms such as fever, cough, sputum production, and breathlessness. However, these symptoms closely resemble those of OPD itself, making the diagnosis complex and challenging [5]. In order to enhance the early diagnostic accuracy of OPD combined with IPA, various experimental methods have been introduced in clinical practice. Detection of IgG antibodies against Aspergillus fumigatus is a method used to diagnose IPA by measuring specific IgG antibody levels in the patient’s blood. Following infection or exposure to Aspergillus fumigatus, the immune system produces specific IgG antibodies to counteract the fungus [6]. Measuring the levels of specific IgG antibodies against Aspergillus fumigatus in the patient’s serum can aid in diagnosing the presence of Aspergillus infection. The advantage of this method lies in its relatively prolonged duration, enabling detection of chronic or recurrent infections of aspergillosis [7]. Similar to IgG detection, IgM antibody testing against Aspergillus fumigatus detects specific IgM antibody levels. IgM antibodies are typically produced in the early stages of infection, making this method valuable for the early diagnosis of infections. While this method can provide earlier detection of aspergillosis, its relatively shorter duration may limit its ability to detect chronic or recurrent infections [8]. Galactomannan (GM) antigen detection in serum is a direct method for detecting cell wall components of Aspergillus fumigatus. The cell wall of Aspergillus fumigatus contains galactomannan, and during an infection of aspergillosis, this antigen may be released into the patient’s bloodstream [9]. By measuring the levels of GM antigen in serum, both early and recurrent infections of aspergillosis can be rapidly diagnosed. The advantage of this method lies in its relatively high sensitivity and specificity, making it valuable in the diagnosis of OPD combined with IPA in patients [10]. Recent studies have demonstrated the diagnostic potential of lateral flow assay (LFA) and GM testing in detecting IPA, with a meta-analysis showing that LFA and GM-LFA have similar diagnostic accuracy, with a sensitivity of 77% and 75%, respectively, and specificity of 88% and 87%, respectively [11]. Furthermore, a systematic review and meta-analysis revealed a high burden of Aspergillus sensitization (AS) and allergic bronchopulmonary aspergillosis (ABPA) in children with asthma, highlighting the need for screening and early diagnosis [12]. In addition, metagenomic next-generation sequencing (mNGS) has emerged as a promising tool for diagnosing IPA, with a study demonstrating its superior diagnostic capability compared to fungal culture and GM testing in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) [13]. A comprehensive review of Aspergillus species distribution and risk factors for aspergillosis in mainland China also underscored the importance of understanding the epidemiology and clinical characteristics of aspergillosis to guide diagnosis and treatment [14].
Aim
This work compared the three aforementioned detection methods, evaluating their diagnostic sensitivity and specificity in OPD patients with concurrent IPA, thus providing a more reliable basis for clinical diagnosis. Additionally, we will explore the RFs linked with OPD combined with IPA, aiming to provide more targeted strategies for early intervention and treatment.
Material and methods
This was a case control study. Seventy-six patients hospitalized in the respiratory department of our hospital due to acute exacerbation of OPD from January 2022 to December 2023 were collected/enrolled. They were categorized into an IPA group (n = 39) and a non-IPA group (n = 37) based on the presence of aspergillus fumigatus infection in the lungs. The research subjects agreed to sign informed consent forms with the consent of their family members, and the implementation of this work obtained approval from the hospital’s ethics committee.
Patients enrolled had to satisfy all the following conditions: age ≥ 18 years; cases meeting the diagnostic criteria for OPD, including confirmed irreversible airflow limitation through pulmonary function tests, with detailed medical records available for review, including clinical manifestations, imaging data, microbiological test results, among others.
Patients with any of following conditions had to be excluded: concomitant immunodeficiency caused by other reasons, such as HIV infection or long-term use of immunosuppressive agents; concomitant active pulmonary infections of other origins, such as bacterial pneumonia and tuberculosis, which may interfere with the diagnosis of IPA; history of IPA; insufficient microbiological or imaging examinations during hospitalization, making it impossible to confirm or rule out IPA; and severe hepatic or renal dysfunction or other significant organ failure, with an expected survival time of < 6 months.
The diagnosis of IPA required the comprehensive assessment of clinical manifestations, imaging examinations, microbiological evidence, and other relevant information. 1) Clinical criteria: presence of typical clinical manifestations, like fever unresponsive to antibiotic treatment, dyspnoea, chest pain, haemoptysis, etc. 2) Imaging criteria: chest computed tomography (CT) scans showing typical IPA signs, including but not limited to “halo sign”, “cavity formation” or “nodules”. 3) Microbiological criteria: direct microscopic examination or culture confirming the presence of Aspergillus in lung tissue or bronchoalveolar lavage fluid (BALF).
Data collection
Retrospective collection of clinical data for all patients, including age, gender, body mass index (BMI), annual frequency of acute exacerbations in OPD, smoking history, comorbidities (hypertension, diabetes, chronic cardiac insurance, chronic renal failure (CRF), cirrhosis), medication history (duration of broad-spectrum antibiotic (BSA) usage, duration of long-term corticosteroid usage), and serum albumin levels.
Laboratory detection methods
The specific information for primary reagents and instruments adopted here were detailed in Table 1.
Table 1
Primary reagents and instruments
Aspergillus fumigatus IgG antibody detection: a 5 ml venous blood sample was collected from the patient, subjected to 15-min centrifugation at 3,000 rpm, and the serum was separated and stored at –80°C for testing. The IgG antibody detection kit was utilized, and all reagents were prepared following the instructions to ensure room temperature for both reagents and samples. The serum sample was introduced to the microplate pre-coated with Aspergillus fumigatus-specific antigen and incubated overnight at 37°C. This facilitated the specific binding of IgG antibodies in the serum to the Aspergillus fumigatus antigen fixed on the plate. Post-incubation, the plate underwent washing at least 3 times with washing buffer to eliminate unbound components. Secondary anti-human IgG antibodies labelled with peroxidase were introduced to each well to detect specifically bound IgG antibodies. Another incubation was conducted for the secondary antibodies to bind to the IgG antibodies, followed by washing to remove unbound secondary antibodies. Subsequently, substrate was incorporated, and the substrate’s reaction with the enzyme caused a visible colour change. Finally, the stop solution was added to terminate the substrate reaction. Readings were obtained by measuring the optical density (OD) value of each well using an enzyme-linked immunosorbent assay (ELISA) reader. The concentration of IgG antibodies in the sample was calculated by comparing the OD value of the sample with the standard curve of known concentrations.
Aspergillus fumigatus IgM antibody detection: the testing process was similar to IgG detection, with the difference being the use of peroxidase-labelled anti-human IgM antibodies to detect specifically bound IgM antibodies. The remaining steps (sample preparation, reagent preparation, sample addition, incubation, washing, substrate addition, reaction termination, and reading) were the same as the IgG detection process.
GM antigen detection: a 5 ml blood sample was collected from the patient’s vein under sterile conditions, centrifuged to separate the serum, and the serum sample was stored at –80°C. Following the GM kit instructions, the serum samples were added to each test well of the microplate. Subsequently, the microplate loaded with samples was incubated at a constant temperature of 37°C, facilitating the specific binding of the GM antigen in the serum to the antibodies fixed on the microplate. After incubation, the microplate was rinsed with washing solution to eliminate non-specifically bound substances. Enzyme-labelled secondary antibodies were added to the washed wells, allowing them to specifically bind to the GM antigen already bound to the fixed antibodies. Afterwards, another incubation and wash cycle was performed, and enzyme substrate was subsequently incorporated to initiate the colour reaction. The enzyme-labelled secondary antibodies reacted with the substrate, inducing a colour change. Finally, the reaction stop solution was introduced to halt the colour reaction. The results were read using an ELISA reader, and the concentration of GM antigen in the sample was determined by comparing the OD values of the sample with a pre-established standard curve.
Judgment criteria
GM antigen detection: a GM index (or OD ratio) ≥ 0.5 is generally considered positive, suggesting the possibility of IPA.
Aspergillus fumigatus IgG antibody detection: typically, a serum IgG antibody concentration below 20 AU/ml is considered negative; IgG antibody concentrations in the borderline range, such as 20–40 AU/ml, were considered suspiciously positive; IgG antibody concentrations above 40 AU/ml suggested positivity, indicating the possibility of infection.
Aspergillus fumigatus IgM antibody detection: a serum IgM antibody concentration below 10 AU/ml was considered negative; that of 10–20 AU/ml may indicate a recent infection and were considered suspiciously positive; and these exceeding 20 AU/ml are considered positive, supporting the diagnosis of a recent or active Aspergillus fumigatus infection.
Statistical analysis
All experimental data were statistically analyzed using SPSS 27.0. Descriptive statistics for continuous variables were displayed as mean ± standard deviation, and categorical variables were given as frequencies. Statistical inferences were made using the χ2 test for categorical data, and independent sample t-tests were adopted for continuous data since the data followed a normal distribution. Factors with statistically significant results in the univariate analysis were chosen as independent variables, with the presence or absence of IPA in OPD patients as the dependent variable. Additionally, logistic regression analysis was employed for the analysis of risk factors (RFs). To prepare the data for multivariate logistic regression (MLR) analysis, adjustments were made to the variables to eliminate potential confounding factors. The variables were assigned as follows based on the univariate analysis: smoking history was assigned as independent variable 1, with yes = 1 and no = 0; history of diabetes was assigned as independent variable 2, with yes = 1 and no = 0; use of BSAs for ≥ 2 weeks was assigned as independent variable 3, with yes = 1 and no = 0; use of corticosteroids for ≥ 3 weeks was assigned as independent variable 4, with yes = 1 and no = 0; and serum albumin levels were assigned as independent variable 5, with levels < 37 g/l = 1 and ≥ 37 g/l = 0. The outcome variable, OPD combined with IPA or not, was assigned as the dependent variable, with yes = 1 and no = 0. P < 0.05 was considered statistically significant.
Results
According to Table 2, the following indicators were observed to have significant effects on the presence of IPA in OPD patients: compared to the non-IPA group, patients in the IPA group were more likely to have a history of smoking (p = 0.012), diabetes (p = 0.001), and use of BSAs for ≥ 2 weeks (p < 0.001) and corticosteroids for ≥ 3 weeks (p < 0.001). Additionally, patients in the IPA group had significantly lower serum albumin levels (30.63 ±3.25 vs. 37.34 ±3.51 g/l, p < 0.001). There were no significant differences between the two groups in terms of age, gender, BMI, frequency of COPD exacerbations, history of hypertension, CHF, CRF, or cirrhosis.
Table 2
Results of univariate analysis
The variables identified as being statistically different through univariate analysis, including smoking history, history of diabetes, use of BSAs for ≥ 2 weeks, use of corticosteroids for ≥ 3 weeks, and serum albumin levels, were incorporated as independent variables into a MLR analysis (Table 3).
Table 3
MLR analysis results
As outlined in Table 4, the use of BSAs for ≥ 2 weeks (p = 0.001), the use of corticosteroids for ≥ 3 weeks (p = 0.003), and serum albumin levels (p = 0.004) were independent RFs for OPD patients with concurrent IPA.
Table 4
Detection results of three detection methods
| Method | TP | FP | TN | FN | Sensitivity | Specificity |
|---|---|---|---|---|---|---|
| GM antigen | 25 | 9 | 28 | 14 | 64.1% | 75.68% |
| Antibody IgG | 24 | 7 | 30 | 15 | 61.54% | 81.08% |
| Antibody IgM | 20 | 10 | 27 | 19 | 51.28% | 72.97% |
Comparison of three detection methods
As demonstrated in Table 4 using 0.5 as the positive threshold for GM antigen detection, there were 25 true positives (TP), 9 false positives (FP), 28 true negatives (TN), and 14 false negatives (FN). The sensitivity and specificity of GM antigen detection were 64.1% and 75.68%, respectively.
For aspergillus fumigatus-specific IgG antibody detection, it identified 24 TP, 7 FP, 30 TN, and 15 FN, yielding the sensitivity and specificity of 61.54% and 81.08%, respectively.
Regarding aspergillus fumigatus-specific IgM antibody detection, 20 TP, 10 FP, 27 TN, and 19 FN were identified, resulting in the sensitivity and specificity of IgM antibody detection of 51.28% and 72.97%, respectively.
Discussion
Our research suggests that prolonged use of BSAs and corticosteroids, as well as low serum albumin levels, are independent risk factors for IPA in asthma or COPD patients. The use of corticosteroids may also be related to the severity of OPD itself, indicating that these patients may already have more severe lung damage and functional impairment, making them more susceptible to developing IPA [15]. Therefore, when using corticosteroids to treat OPD, physicians need to assess the necessity of long-term use and the potential risk of IPA. For patients who must use corticosteroids for an extended period, regular monitoring and proactive prevention and intervention measures are advisable.
Our findings suggest that low serum albumin levels, indicative of poor nutritional status and/or chronic inflammation, combined with prolonged use of broad-spectrum antibiotics and corticosteroids, are independent risk factors for invasive pulmonary aspergillosis in outpatients. Gu et al. [16] planned to develop and validate a risk model to predict the occurrence of IPA in hospitalized patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD). They randomly assigned 880 AECOPD patients into training and validation sets, developed a nomogram model using multivariate logistic regression in the training set, and internally validated its discriminatory and calibration performance. The results signified that the model included independent factors associated with IPA, such as GOLD III–IV level of lung function, using BSAs for more than 10 days in the past month, total corticosteroid dose (oral or intravenous) exceeding 265 mg in the past 3 months, and serum albumin levels below 30 g/l. In the validation set, the model exhibited good discriminatory and calibration abilities, and decision curve analysis suggested its practical application in clinical settings. In comparison to our study, their study provides a quantifiable risk assessment tool focused on early identification of IPA risk in AECOPD patients in clinical practice.
In contrast to the study by Palanivel et al. in 2024 [17], which focused on the prevalence and risk factors for chronic pulmonary aspergillosis (CPA) in COPD patients with acute exacerbations, our study investigated the sensitivity of serum galactomannan antigen in diagnosing IPA in patients with obstructive pulmonary disease. While Palanivel et al. reported a prevalence of CPA of 9.8% among COPD patients with acute exacerbations, our study found that prolonged use of broad-spectrum antibiotics and corticosteroids, along with low albumin levels, were independent risk factors for concurrent IPA in OPD patients. Interestingly, the sensitivity of serum galactomannan antigen detection in our study (64.1%) was higher than the sensitivity of Aspergillus-specific IgG and IgM antibody detection, which is consistent with the need for early recognition and targeted management of IPA in OPD patients.
Our study identified prolonged use of broad-spectrum antibiotics and corticosteroids, as well as low albumin levels, as independent risk factors for concurrent IPA in patients with obstructive pulmonary disease. In contrast, a previous study by Molinos-Castro et al. [18] found that home oxygen therapy, bronchiectasis, hospital admission in the previous 3 months, and antifungal therapy against Candida spp. in the previous month were associated with a higher risk of pulmonary aspergillosis in patients with COPD. While there is some overlap in the patient population, our study focused on acute exacerbations of obstructive pulmonary disease, whereas the Molinos-Castro et al. study included patients with COPD who had Aspergillus spp. isolated from respiratory samples. The differences in risk factors may be due to the distinct patient populations and study designs. Nevertheless, both studies highlight the importance of identifying risk factors for pulmonary aspergillosis in patients with obstructive pulmonary disease to facilitate early diagnosis and treatment. Additionally, our study demonstrated that serum galactomannan antigen detection has a relatively high sensitivity (64.1%) for diagnosing IPA, which may be a useful tool in conjunction with clinical risk factors to guide diagnosis and treatment.
A study by Tirelli et al. in 2023 investigated the role of Aspergillus-specific IgE, IgG, and IgG4 in diagnosing IPA [19]. By prospectively storing and testing 447 serum samples from suspected IPA patients, the study found that confirmed IPA patients diagnosed through BAL culture and/or GM antigen detection had higher levels of specific Aspergillus IgG in their serum. Notably, Aspergillus fumigatus-specific IgG at a cutoff of 22.6 mgA/l exhibited the highest sensitivity. Simultaneously, a combination of lower levels of specific Aspergillus IgE, IgG, and IgG4 below the cutoff values showed over 90% of negative predictive values for both BAL culture and GM antigen detection. Thus, the study proposed that the detection of Aspergillus-specific immunoglobulins could serve as a preliminary screening tool for suspected IPA patients, complementing more invasive detection methods. In contrast, the focus of this study is on evaluating the sensitivity of GM antigen detection in diagnosing IPA in OPD patients, while the study by Tirelli et al. [19] concentrates on assessing the potential application of Aspergillus-specific immunoglobulins in preliminary IPA screening. Both studies involve the application of serum biomarkers in IPA diagnosis, but with different emphases and methodologies. This study emphasizes GM antigen detection, whereas Tirelli et al. explored the combined measurement of various immunoglobulins. Their research suggests that when these immunoglobulin levels fall below specific cutoff points, they can serve as robust negative predictive indicators for IPA, potentially offering a non-invasive method for IPA screening in OPD patients.
On the other hand, this work was subjected to several limitations. Firstly, the sample size may be insufficient, and larger sample sizes are preferable, especially for statistical analyses such as MLR analysis, as they can provide stronger statistical power, leading to more reliable conclusions. Additionally, the work based on cross-sectional data, which could not establish causation and can only provide evidence of associations. Moreover, the research was conducted at a single centre, limiting the generalizability of the results, as they may not be widely applicable to different geographical locations and populations. The sensitivity and specificity of the detection methods may be influenced by factors such as the timing of the tests, individual patient differences, and variations in reagent batches, potentially leading to fluctuations in results. Furthermore, there might be other factors not considered in the study, such as specific immune status and concurrent medication use by patients, which could affect the development of IPA and its detection. Future research should aim to address these limitations by expanding the sample size and conducting studies at multiple medical centres in different regions to enhance the generalizability and reliability of the findings. Longitudinal tracking of OPD patients over time would provide a better understanding of the developmental process and risk factors for IPA. There is a hope to develop more sensitive and specific biomarker detection methods for IPA to improve diagnostic accuracy.
Conclusions
This work provided a detailed analysis of OPD patients with concomitant IPA, revealing that long-term use of broad-spectrum antibiotics (≥ 2 weeks), prolonged use of corticosteroids (≥ 3 weeks), and low serum albumin levels were independent risk factors for combined IPA. Among the three different IPA detection methods, GM antigen detection signified slightly higher sensitivity than aspergillus-specific antibodies IgG and IgM detection. However, none of these detection methods achieved the desired sensitivity, indicating that in actual clinical practice, a single detection method may be insufficient for diagnosing IPA. A comprehensive approach involving clinical symptoms, imaging studies, and multiple biomarker detections was necessary for accurate diagnosis. This work emphasized the importance of identifying and assessing the risk of combined IPA in OPD (asthma and COPD) patients. It suggested that caution was needed in the clinical use of antibiotics and steroids, with increased attention to the nutritional and immune status of patients.
