A total of 4705 patients underwent peripheral blood mNGS testing at 4 hospitals, Zhejiang, between 01/02/2021 and 30/12/2022. Patients who were found to have detectable nucleic acid of the Aspergillus genus were included. Clinical comprehensive diagnoses of invasive aspergillosis or non-infection were made by three senior experts in respiratory medicine, infectious diseases, and radiology. This decision was reached through discussions with the patient’s primary medical team, considering clinical symptoms, laboratory results, imaging studies, microbiology, pathology examinations, as well as treatment, prognosis, etc. Cases with incomplete clinical data, individuals under 18 years old, pregnant or postpartum women, and cases of non-hospitalized patients were excluded.

IA is categorized into confirmed, probable and possible diagnosis. Cases included in this study had at least one instance of blood mNGS detecting Aspergillus, upon which the diagnosis was based. The diagnostic criteria followed the guidelines for invasive fungal diseases jointly published by the European Organization for Research on Treatment of Cancer (EORTC) and the Mycoses Study Group Education and Research Consortium (MSGERC) in 2020 (Pappas et al., 2021). Patients were categorized into confirmed, probable, and possible diagnoses of IA based on these diagnostic criteria.

Confirmed Diagnosis: Criteria: Pathological findings from surgical or biopsy tissue at the infection site confirm Aspergillus infection.

Probable Diagnosis: Host factors, clinical symptoms or signs, and microbiological criteria consistent with Aspergillus infection are required. The microbiological criteria for clinical diagnosis are as follows: Aspergillus identified by smear or culture from specimens at the infection site, and detection of the same Aspergillus species in at least two mNGS tests (including blood or specimens from the infection site).

Possible Diagnosis: Host factors and clinical features consistent with Aspergillus infection. Host factors include hematological malignancies, solid organ and hematopoietic stem cell transplantation (HSCT), chemotherapy or immunotherapy for solid tumors, granulocyte deficiency, prolonged use of corticosteroids (average minimum dose exceeding 0.3 mg/kg/day for more than 3 weeks of prednisone or equivalent), ICU critically ill patients, chronic obstructive pulmonary disease, diabetes, liver failure, renal failure, etc (Gaffney et al., 2023).

The diagnostic methods for Aspergillus spp. include fungal culture, calcofluor white stain for fungi, galactomannan (GM) antigen test. The GM antigen was detected using a Platelia Aspergillus EIA kit (Bio-Rad, Marnes-la-Coquette, France), according to the manufacturer’s instructions. Both positive and negative controls were included in each assay. A result with an index value of >0.5 (serum) or 0.8 (BALF) in duplicate tests was considered positive.

The testing protocol, experimental parameters, quality control, and test result reporting of the plasma mNGS testing in our laboratory are described by Han et al. (7) Briefly, peripheral venous blood samples were collected and sent to the clinical laboratory as soon as possible. Plasma was separated under the centrifugal parameters of 500 g for 5 min at 4°C. The separated plasma is used to extract cell free DNA (cfDNA) and cell free RNA (cfRNA) and libraries were constructed. The size distribution was measured and the concentration of the libraries was quantified. Library pools were then loaded onto the Illumina Nextseq CN500 sequencer for 50 cycles of single-end sequencing (SE-50), generating approximately 20 million reads for each library. Microbial reads were aligned to the database using Burrows-Wheeler Aligner software which contains a proprietary curated database consisting of more than 20,000 microbial reference genomes. Most mNGS results returned within 36 hours.

Demographic information, risk factors, underlying diseases, use of immunosuppressive drugs, steroids, chemotherapy drugs in the month preceding onset, clinical symptoms, laboratory examinations, imaging data, as well as treatment and prognosis, etc., were collected for the 334 included patients.

Statistical comparisons were made between patients with positive blood mNGS for IA and contaminated patients. General conditions, risk factors, clinical manifestations, and prognosis were compared.

SPSS 27.0 software (IBM Corporation, Chicago, Illinois, USA) was used for data analysis. Continuous variables conforming to a normal distribution were expressed as mean ± standard deviation (x ± s), and an independent sample t-test was used for intergroup comparison of measurement data. Non-normally distributed continuous variables were expressed as median and interquartile range, and the Mann-Whitney U test was used for comparison. Count data were compared using the chi-square test or Fisher’s exact test. A P value < 0.05 indicated a statistically significant difference.

A total of 334 patients were included, comprising 188 males (56.3%) and 146 females (43.7%), with a median age of 58 years (range 43 to 68). Underlying diseases included hematological malignancies in 202 cases, with 128 cases of leukemia (38.3%), 34 cases of lymphoma (10.2%), 26 cases of myelodysplastic syndrome (7.8%), and 14 cases of multiple myeloma (4.2%). There were 54 cases post-hematopoietic stem cell transplantation (16.2%), 36 cases post-solid organ transplantation (10.8%), and 44 cases of diabetes (13.2%). Other underlying conditions included 32 cases of solid tumors (9.6%), among others, Table 1. Overall clinical and pathogen diagnosis of the recruited patients are showed in Figure 1.

There were 4 patients with confirmed invasive pulmonary aspergillosis based on histopathological evidence. There were 2 men and 2 women, 2 patients with lung transplantation, 2 patients with hematological malignancies, 1 patient with diabetes and 1 patient with bronchiectasis. Four patients were further confirmed to have Aspergillus infection through bronchoscopy biopsy and histopathological examination. In addition, both patients were confirmed to have Aspergillus infection in bronchoalveolar lavage fluid culture and mNGS testing. The Aspergillus infection in all 4 patients improved after treatment.

There were 62 cases, including 32males, aged 21-95 (56.1 ± 16.7) years. Among them, 58 cases were clinically diagnosed with invasive pulmonary aspergillosis, 4 case with disseminated invasive aspergillosis, and 4 case with urinary system Aspergillus infection. Underlying diseases included 44 cases of leukemia, 4 cases of lymphoma, 4 cases of myelodysplastic syndrome, 2 case of multiple myeloma, 10 cases after solid organ transplantation, 12 cases after hematopoietic stem cell transplantation, 4 cases of diabetes, 2 case of chronic obstructive pulmonary disease, and 6 cases of solid tumors. Nine patients had the same genus Aspergillus detected in 2–4 peripheral blood mNGS tests with an interval of 16 (10, 61) days. 20 cases were positive in culture and smear. 24 cases had the same genus of Aspergillus detected by using mNGS in sputum, lavage fluid, or cerebrospinal fluid. Among the 62 cases, there were 28 cases of A. fumigatus, 24 cases of A. flavus, 2 case of A. terreus, 2 case of A. oryzae, 2 case of A. niger, 2 case of A. clavatus, and 2 case of A. ustus. Pulmonary CT of 58 IPA patients showed imaging features consistent with Aspergillus infection, including nodular shadows, patchy shadows, halo signs, cavities, consolidation, etc. After active treatment, 36 cases improved, and 26 cases died, 28d mortality: 41.9%.

There were 134 suspected cases, including 84 males (62.7%). These patients, in addition to one instance of blood mNGS detecting Aspergillus, also presented with both the risk factors for Aspergillus infection and clinical features consistent with Aspergillus infection. Clinical characteristics are detailed in Table 2.

There were 134 contaminated cases, including 70 males (52.2%). The age ranged from 18 to 90 years (mean ± SD, 51.3 ± 17.9). Clinical characteristics are detailed in Table 3.

Only Aspergillus was detected in the plasma of 84 patients, and other microorganisms were detected in the plasma of 250 patients. The combined detected microorganisms are listed in Table 4. In IA group and false positive group, there was no significant difference in the detection of pathogens other than Aspergillus spp.

A comparison of clinical data between 200 cases of blood mNGS-positive invasive aspergillosis and 134 contaminated cases revealed that the infection group had a higher incidence of lung imaging consolidation, masses, and cavities. There was no statistically significant difference in the read counts of Aspergillus between the infection group and the false positive group. Patients in the infection group had lower platelet counts, higher CRP levels, more critical cases, and poorer prognosis. Table 5.