Metagenomic Next-Generation Sequencing for Pulmonary Fungal Infection Diagnosis: Lung Biopsy versus Bronchoalveolar Lavage Fluid

Figures & data

Table 1 Patient Demographic Characteristics

Characteristics		Patient, N (%)
Age		43.2±18.5
Sex	Male	76 (71.7%)
	Female	30 (28.3%)
Underlying disease	Immunocompromised	84 (79.3%)
	Non-Immunocompromised	22 (20.8%)

Table

Patient ID	Sex	Age/yr	Underlying Disease	Immunocompromised	Pathology Results	Culture Results	GM Test	mNGS (Biopsy) Based Diagnosis	mNGS (BAL) Based Diagnosis	Final Clinical Diagnosis
1	Male	46	ALL	Yes	Pink amorphous substance, negative for PAS, hexamine silver and acid-fast staining	Mold growth	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
2	Male	19	ALL	Yes	Alveolar septal fibrous tissue hyperplasia	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
3	Male	43	AML	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
4	Male	19	T lymphoblastic acute leukemia	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
5	Male	47	No	No	Alveolar septal fibrous tissue hyperplasia with lymphocyte infiltration	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
6	Male	63	No	No	Interstitial fibrous tissue hyperplasia, scattered lymphocyte infiltration, alveolar epithelial hyperplasia, fibroblast thrombosis in the alveolar cavity	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
7	Female	14	ALL	Yes	Alveolar septal fibrous tissue hyperplasia with lymphocyte infiltration	Viridans streptococci, Micrococcus pharyngis	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
8	Male	11	Hodgkin’s Lymphoma	Yes	Chronic inflammatory cell infiltration in the alveolar compartment,	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
9	Female	39	Mixed connective tissue disease	Yes	Alveolar septal fibrous tissue hyperplasia with lymphocyte infiltration	Streptococcus pneumoniae	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
10	Male	15	ALL	Yes	Alveolar septal fibrous tissue mild hyperplasia and chronic inflammatory cell infiltration,	Negative	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
11	Male	25	ALL	Yes	Interstitial lymphocytes, plasma cell infiltration, neutrophils, fibrinous exudate	Citrobacter koseri	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
12	Male	68	Pancytopenia	Yes	No	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
13	Female	51	Rheumatoid Arthritis	Yes	Fibrous tissue hyperplasia and lymphoid tissue hyperplasia,	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
14	Male	39	AML	Yes	Widened alveolar space, fibrous tissue hyperplasia, focal alveolar hyperplasia, Scattered lymphocytes and tissue cells infiltration, focal carbon dust deposition	Viridans streptococci, Neisseria sicca	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
15	Male	52	ALL	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
16	Female	30	Chronic gastritis	No	Alveolar septal fibrous tissue hyperplasia, Slurry cellulosic exudate	Citrobacter koseri	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
17	Male	32	AML	Yes	Tumorous lesions to be excluded	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
18	Male	56	ALL	Yes	Mild hyperplasia of alveolar septal fibrous tissue, focal type II alveolar epithelial hyperplasia	Viridans streptococci, Neisseria sicca	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
19	Male	22	ALL	Yes	Necrotic tissue	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
20	Male	18	Acute B lymphocytic lymphoma	Yes	Mild hyperplasia of alveolar septum fibrous tissue	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
21	Female	34	No	No	Mild hyperplasia of alveolar septum fibrous tissue	Viridans streptococci, Staphylococcus epidermidis	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
22	Male	58	MDS	Yes	Alveolar septal fibrous tissue proliferates, scattered lymphocytes infiltrate, fibroblast thrombus formation in the alveolar cavity,	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
23	Male	68	AML	Yes	Alveolar septal fibrous tissue hyperplasia with chronic inflammatory cell infiltration, local alveolar epithelial hyperplasia,	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
24	Male	22	ALL	Yes	No	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
25	Female	55	AML	Yes	Alveolar septum widening, interstitial fibrous tissue hyperplasia with inflammatory cell infiltration	Acinetobacter baumannii complex	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
26	Male	48	AML	Yes	No	Viridans streptococci	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
27	Female	63	AML	Yes	Mold hyphae	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
28	Male	60	AML	Yes	Mucosal lamina propria fibrosis with medium to small nuclei and clusters of cells stained with cytoplasm, not supporting myeloid leukemia cells	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
29	Male	47	ALL	Yes	Alveolar septal fibrous tissue hyperplasia, Foamy cell aggregation, No leukemia cells seen	Viridans streptococci	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
30	Male	25	ALL	Yes	Inflammatory exudate and coagulated necrotic tissue	Meningeal sepsis Eliza Platinum bacteria, Citrobacter koseri	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
31	Female	15	CML	Yes	Coagulating necrotic tissue and very little fibrous tissue	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
32	Male	58	AML	Yes	No tumor cells seen	Pseudomonas putida, Stenotrophomonas maltophilia	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
33	Male	18	ALL	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
34	Male	54	No	No	Chronic inflammation of mucosa	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
35	Male	71	MarginalzoneB-cell lymphoma	Yes	Chronic inflammatory cell infiltration in alveolar space	Viridans Streptococci	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
36	Female	41	No	No	Fibroblast thrombus formation in the alveolar cavity	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
37	Female	42	Acute Laryngitis	No	Fibrous tissue hyperplasia and lymphocyte infiltration	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
38	Female	63	CML	Yes	Fibrous tissue hyperplasia, with scattered inflammatory cell infiltration	Enterobacter cloacae	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
39	Male	22	ALL	Yes	Interstitial fibrous tissue hyperplasia with inflammatory cell infiltration	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
40	Male	38	ALL	Yes	Alveolar septum widening, interstitial fibrous tissue hyperplasia	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
41	Female	31	AML	Yes	Legion of Fungi	Enterobacter cloacae	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
42	Female	64	Postoperative Breast Tumor	No	Chronic inflammatory cell infiltration with mild fibrous tissue hyperplasia	Viridans Streptococci	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
43	Male	42	No	No	Fibroblast thrombus formation in the alveolar cavity	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
44	Male	49	AML	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
45	Male	47	Acute leukemia	Yes	A lot of fungus and a little inflammatory exudate	Staphylococcus aureus	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
46	Female	49	ALL	Yes	No	Negative	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
47	Male	64	AML	Yes	Mild proliferation of alveolar septal fibrous tissue with a little lymphocyte infiltration	Viridans streptococci	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
48	Male	38	Immunorelated pancytopenia	Yes	Alveolar hemorrhage, edema, and hemosiderin deposition	Negative	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
49	Female	43	Asthma	No	Fibroblast plug formation	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
50	Male	51	AML	Yes	No	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
51	Male	61	Diabetes, coronary heart disease	No	Legion of fungi. methenamine silver stain +.	Negative	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
52	Male	23	ALL	Yes	Non-neoplastic lesions	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
53	Male	66	Hypertension	No	Fibrous tissue hyperplasia and inflammatory cell infiltration of alveolar septum	Micrococcus pharyngis	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
54	Male	61	Chronic obstructive pulmonary disease, hypertension	No	Non-small cell carcinoma, predisposing to squamous cell carcinoma	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
55	Male	49	Acute nonlymphocytic leukemia	Yes	Powdery cellulose-like exudate, neutrophils, degenerated and necrotic cells	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
56	Male	51	AML	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
57	Male	30	Aplastic anemia	Yes	Alveolar septal fibrous tissue hyperplasia with lymphocyte infiltration	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
58	Female	46	Hypertension	No	Alveolar septum widening, interstitial fibrous tissue proliferation and inflammatory cell infiltration	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Cryptococcal infection
59	Male	47	Aplastic anemia	Yes	Mold clusters, methenamine silver stain +	Klebsiella pneumoniae	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
60	Male	65	Hypertension, diabetes, coronary heart disease	No	Lymphocyte infiltration, acute and chronic inflammatory cell infiltration	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
61	Male	72	Lung tumor	Yes	Lymphocyte infiltration, alveolar septal fibrous tissue hyperplasia	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
62	Male	11	Acute mixed cell leukemia	Yes	Acute and chronic inflammatory cell infiltration	Viridans streptococci	Positive	Exclude pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
63	Female	64	Diabetes, nodular goiter	No	Alveolar septal fibrous tissue hyperplasia with chronic inflammatory cell infiltration	Escherichia Coli	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
64	Male	75	Acute leukemia	Yes	Chronic inflammation	Staphylococcus epidermidis	Positive	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
65	Male	73	Acute monocytic leukemia	Yes	Inflammatory cell infiltration	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
66	Female	33	AML	Yes	Acute and chronic inflammatory cell infiltration, virus infection not excluded	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
67	Female	18	ALL	Yes	Alveolar epithelial hyperplasia, focal fibroblast thrombus formation, mild hyperplasia of alveolar septal fibrous tissue, and organizing lesions not excluded	Negative	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
68	Male	44	T-cell acute lymphoblastic leukemia	Yes	No	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
69	Female	10	Aplastic anemia	Yes	Mesenchymal tissue and large amount of acute exudate	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
70	Male	38	No	No	Lymphocyte infiltration of alveolar septum with fibrous tissue hyperplasia, fibroblast plug	Micrococcus pharyngis	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
71	Female	39	Leukemia	Yes	Alveolar septal fibrous tissue hyperplasia with chronic inflammatory cell infiltration	Negative	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
72	Male	70	Diabetes, alcoholic cirrhosis	No	No	Viridans streptococci	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
73	Male	21	AML	Yes	No tumor cells	Negative	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
74	Male	38	Immunorelated pancytopenia	Yes	Chronic inflammation of the mucosa	Enterococcus faecalis	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
75	Male	13	ALL	Yes	Legion of Fungi	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
76	Male	69	No	No	Chronic inflammation of mucosa	Viridans streptococci	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
77	Male	66	Hypertension	No	Proliferation of interstitial fibrous tissue and lymphoid tissue with scattered inflammatory cell infiltration,	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
78	Male	23	Acute leukemia	Yes	No tumor cells	Viridans streptococci	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
79	Male	58	ALL	Yes	Amorphous tissue, small airway mucosa with chronic inflammatory cell infiltration and fibrous tissue proliferation	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
80	Male	31	AML	Yes	No	Viridans streptococci	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
81	Male	53	MDS	Yes	Alveolar septal fibrous tissue hyperplasia and local fibroblast thrombus formation.	Negative	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
82	Male	17	AML	Yes	No	Candida albicans	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
83	Male	29	Aplastic anemia	Yes	No	Achromobacter xylosoxidans	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
84	Male	43	ALL	Yes	Suspicious fungus	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
85	Female	9	AML	Yes	No	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
86	Male	53	AML	Yes	No tumor cells seen	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
87	Male	66	No	No	Proliferation of interstitial fibrous tissue with scattered inflammatory cell infiltration, fibroblast plugs,	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
88	Male	50	Hypertension	No	Interstitial fibrous tissue with inflammatory cell infiltration,	Viridans streptococci	Negative	Pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
89	Female	59	Leukemia	Yes	No	C. glabrata, viridans streptococci	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
90	Female	56	AML	Yes	Mild hyperplasia of alveolar septum fibrous tissue	Viridans streptococci	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
91	Male	48	Lymphoma, hypertension	Yes	Fibrinous exudative necrosis, a small amount of inflammatory cells and epithelial cells	Mold growth	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
92	Female	36	AML	Yes	Alveolar septal fibrous tissue hyperplasia	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
93	Male	35	Diffuse Large B Cell Lymphoma	Yes	Mild hyperplasia of alveolar septum fibrous tissue	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
94	Male	53	AML	Yes	Alveolar septal fibrous tissue hyperplasia	Negative	Positive	Exclude pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
95	Female	39	ALL	Yes	Alveolar septal fibrous tissue hyperplasia, foam-like macrophage aggregation, type II alveolar epithelial hyperplasia	Viridans streptococci, staphylococcus epidermidis	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Exclude pulmonary fungal infection
96	Male	37	MDS	Yes	Legion of fungi. methenamine silver stain +	Pseudomonas aeruginosa	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
97	Male	26	AML	Yes	Alveolar septal hyperplasia, interstitial fibrous tissue hyperplasia with a small amount of inflammatory cell infiltration	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
98	Male	77	Chronic Lymphocytic Leukemia	Yes	Granulomatous lesions	Negative	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection
99	Male	25	ALL	Yes	Immunohistochemical staining excludes leukemia involvement	Viridans streptococci, Pneumocystis carinii	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
100	Male	73	Chronic Myelocytic Leukemia	Yes	Amorphous necrotic tissue with a small amount of neutrophil infiltration	Viridans streptococci, corynebacterium	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
101	Female	16	ALL	Yes	Cellulose exudation and foam cells, lymphocyte infiltration and fibrous tissue proliferation	Mold growth	Negative	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
102	Female	61	SLE	Yes	Methenamine silver stain +, cryptococcosis	Negative	Positive	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
103	Female	49	SLE	Yes	No	Mold growth	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
104	Male	15	AML	Yes	Extruded peripheral lung tissue, individual alveolar cavity expansion	Negative	Negative	Exclude pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection
105	Male	74	Autoimmune hemolytic anemia	Yes	Fungal hyphae and spores, PAS-positive and methenamine silver	Negative	Positive	Exclude pulmonary fungal infection	Exclude pulmonary fungal infection	Pulmonary fungal infection
106	Female	21	ALL	Yes	Silk-like structure, histochemical staining shows PAS positive, mold hyphae	Negative	Negative	Pulmonary fungal infection	Pulmonary fungal infection	Pulmonary fungal infection

Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphocytic leukemia; MDS, myelodysplastic syndrome.

Figure 1 Methods used to diagnose pulmonary fungal infections.

Table 3 Performance of mNGS and the Conventional Test in the Diagnosis of Pulmonary Fungal Infections

	Sensitivity % (95% CI)	Specificity % (95% CI)	PPV % (95% CI)	NPV % (95% CI)	P value	P value
mNGS (biopsy)	80.0^a,b (65.0–89.9)	91.8^A,B (81.2–96.9)	87.8 (73.0–95.4)	86.2 (74.8–93.1)	0.754^a	0.388^A
mNGS (balf)	84.4^a,c (69.9–93.0)	85.3^A,C (73.3–92.6)	80.9 (66.3–90.4)	88.1 (76.5–94.7)	0.002^b	0.774^B
Conventional test	44.44^b,c (30.0–59.9)	88.52^B,C (77.2–94.9)	74.1 (53.4–88.1)	68.4 (56.8–78.1)	0.0003^c	0.774^C

Notes: Sensitivity: a, biopsy-mNGS vs BALF-mNGS; b, biopsy-mNGS vs conventional test; c, BALF-mNGS vs conventional test. Specificity: A, biopsy-mNGS vs BALF-mNGS; B, biopsy-mNGS vs conventional test; C, BALF-mNGS vs conventional test.

Abbreviations: PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.

Table 4 Comparison of the ROC for mNGS and the Conventional Tests

	Area Under Curve	95% Confidence Interval	P value
Biopsy combined balf	0.929	0.862–0.970	P<0.0001
Biopsy	0.8663	0.8122–0.9605	P<0.0001
Balf	0.8632	0.7879–0.9385	P<0.0001

Table 5 Comparison of the Difference in the AUC

	Difference Between Areas	P value
Biopsy combined balf~ biopsy	0.0423	0.0509
Biopsy combined balf ~ balf	0.0654	0.018
Biopsy ~ balf	0.0231	0.5748

Figure 2 Pairwise comparison of the ROC curves.

Abbreviation: ROC, receiver operating characteristic.

Figure 3 (A) Fungi detected using lung biopsy-mNGS. (B) Fungi detected using BALF-mNGS.

Abbreviations: BALF, bronchoalveolar lavage fluid; mNGS, metagenomic next-generation sequencing.

Figure 4 Consistency of the two specimens for mNGS in diagnosing pulmonary fungal infections. The pie chart shows the positive distribution of 106 cases investigated for pulmonary fungal infections using lung biopsy and BALF for mNGS. Among the patients whose mNGS results matched for both specimens, the mNGS results of nine patients showed partial matches, 24 patients showed complete matches, but 54 patients had false-positive results.

Abbreviations: BALF, bronchoalveolar lavage fluid; mNGS, metagenomic next-generation sequencing.

Table 6 Results of the 32 Patients with Matching mNGS Results

Patient ID	mNGS (Biopsy, Fungus Detected)	mNGS (Balf, Fungus Detected)	Final Clinical Diagnosis	mNGS (Biopsy) /mNGS (BAL)	Matching Level
1	Rhizopus microsporus (1337) Aspergillus oryzae (3)	Rhizopus microsporus (23)	Pulmonary fungal infections	58	Partial match
11	Aspergillus oryzae (1257) Aspergillus flavus (1146)	Aspergillus oryzae (4) Aspergillus flavus (1)	Pulmonary fungal infections	314	Complete match
16	Scedosporium (4)	Scedosporium (127)	Pulmonary fungal infections	0.03	Complete match
24	Aspergillus oryzae (155) Aspergillus flavus (102)	Aspergillus oryzae (11) Aspergillus flavus (6)	Pulmonary fungal infections	14	Complete match
27	Aspergillus nidulans (279,595) Rhizomucor pusillus (3861) Aspergillus versicolor (183) Aspergillus calidoustus (175) Aspergillus ustus (137)	Aspergillus nidulans (234) Rhizomucor pusillus (6)	Pulmonary fungal infections	1195	Partial match
29	Aspergillus fumigatus (922) Aspergillus fischer (5)	Aspergillus fumigatus (207)	Pulmonary fungal infections	4	Partial match
31	Rhizomucor pusillus (5320)	Rhizomucor pusillus (2)	Pulmonary fungal infections	2660	Complete match
41	Aspergillus oryzae (863) Aspergillus flavus (207)	Aspergillus oryzae (9)	Pulmonary fungal infections	96	Partial match
45	Coprinopsis cinerea (1380)	Coprinopsis cinerea (49)	Pulmonary fungal infections	28	Complete match
51	Aspergillus fumigatus (41,388)	Aspergillus fumigatus (74)	Pulmonary fungal infections	559	Complete match
52	Pneumocystis jirovecii (12,496)	Pneumocystis jirovecii (6566)	Pulmonary fungal infections	2	Complete match
55	Aspergillus oryzae (46)	Aspergillus oryzae (4) Aspergillus (5)	Pulmonary fungal infections	12	Partial match
57	Pneumocystis jirovecii (360)	Pneumocystis jirovecii (452)	Pulmonary fungal infections	0.8	Complete match
59	Rhizopus microsporus (522,197) Mucor (5755)	Rhizopus microsporus (159)	Pulmonary fungal infections	3284	Partial match
65	Aspergillus (4)	Aspergillus (7)	Pulmonary fungal infections	0.6	Complete match
66	Aspergillus (3)	Aspergillus flavus (13)	Pulmonary fungal infections	0.2	Complete match
68	Rhizopus microsporus (299,937) Mucor (8925) Syncephalastrum (21)	Rhizopus microsporus (352) Mucor (8) Aspergillus (5)	Pulmonary fungal infections	852	Partial match
75	Aspergillus fumigatus (5938)	Aspergillus fumigatus (1178)	Pulmonary fungal infections	5	Complete match
78	Aspergillus flavus (2)	Aspergillus flavus (3)	Pulmonary fungal infections	0.7	Complete match
79	Aspergillus oryzae (150)	Aspergillus oryzae (21) Aspergillus (32)	Pulmonary fungal infections	7	Partial match
80	Lichtheimia ramosa (196) Rhizomucor pusillus (2)	Lichtheimia ramosa (166) Rhizomucor pusillus (39)	Pulmonary fungal infections	1.2	Complete match
82	Aspergillus flavus (488)	Aspergillus (123)	Pulmonary fungal infections	4	Complete match
83	Lichtheimia ramosa (186)	Lichtheimia ramosa (149)	Pulmonary fungal infections	1.2	Complete match
84	Rhizomucor pusillus (880)	Rhizomucor pusillus (9)	Pulmonary fungal infections	98	Complete match
93	Pneumocystis jirovecii (321)	Pneumocystis jirovecii (4)	Pulmonary fungal infections	80.2	Complete match
96	Aspergillus oryzae (1618) Aspergillus flavus (1344)	Aspergillus oryzae (330) Aspergillus flavus (266)	Pulmonary fungal infections	4.9	Complete match
97	Pneumocystis jirovecii (161,687)	Pneumocystis jirovecii (12,504)	Pulmonary fungal infections	12.9	Complete match
99	Pneumocystis jirovecii (14,006)	Pneumocystis jirovecii (2160)	Pulmonary fungal infections	6.5	Complete match
100	Rhizopus oryzae (42)	Rhizopus oryzae (2)	Pulmonary fungal infections	21	Complete match
102	Cryptococcusmans (557)	Cryptococcusmans (12)	Pulmonary fungal infections	46.4	Complete match
103	Aspergillus fumigatus (28)	Aspergillus fumigatus (20)	Pulmonary fungal infections	1.4	Complete match
106	Aspergillus oryzae (1182)	Aspergillus oryzae (48) Pneumocystis jirovecii (6)	Pulmonary fungal infections	24.6	Partial match

Abbreviation: mNGS/mNGS (BALF), mNGS (Biopsy) most detected fungal reads/mNGS (BAL) most detected fungal reads.

Figure 5 Comparison of the fungal reads detected by lung biopsy and BALF for mNGS. Multiple interval: mNGS (Biopsy) most detected fungal reads/mNGS (BAL) most detected fungal reads.

Abbreviations: BALF, bronchoalveolar lavage fluid; mNGS, metagenomic next-generation sequencing.

Figure 6 Comparison of the fungal reads detected by mNGS with matching results from both specimens in 32 patients.

Abbreviation: mNGS, metagenomic next-generation sequencing.

Table 7 Sequencing Results for 22 Patients with Inconsistent Lung Biopsy-mNGS and BALF-mNGS

Patient ID	mNGS (Biopsy, Fungus Detected)	mNGS (BALF, Fungus Detected)	Final Clinical Diagnosis
12	Negative	Candida albicans (96)	Exclude pulmonary fungal infection
14	Negative	Aspergillus fumigatus (201)	Pulmonary fungal infection
26	Rhizopus microsporus (476)	Negative	Pulmonary fungal infection
30	Rhizopus microsporus (3)	Negative	Pulmonary fungal infection
35	Aspergillus (3)	Negative	Exclude pulmonary fungal infection
42	Pneumocystis jirovecii (13)	Negative	Exclude pulmonary fungal infection
46	Aspergillus (5)	Negative	Pulmonary fungal infection
48	Negative	Pneumocystis jirovecii (7)	Exclude pulmonary fungal infection
58	Negative	Pneumocystis jirovecii (2)	Pulmonary fungal infection
62	Negative	Aspergillus (4)	Pulmonary fungal infection
64	Negative	Candida albicans (13) Aspergillus (5)	Exclude pulmonary fungal infection
67	Negative	Aspergillus (3)	Pulmonary fungal infection
71	Aspergillus (3)	Negative	Exclude pulmonary fungal infection
73	Negative	Pneumocystis jirovecii (20)	Exclude pulmonary fungal infection
76	Aspergillus (4)	Negative	Pulmonary fungal infection
81	Negative	Candida tropicalis (6)	Exclude pulmonary fungal infection
87	Negative	Aspergillus (3)	Exclude pulmonary fungal infection
88	Aspergillus (3)	Negative	Exclude pulmonary fungal infection
89	Negative	Candida glabrata (146)	Exclude pulmonary fungal infection
94	Negative	Aspergillus fumigatus (4)	Pulmonary fungal infection
95	Negative	Pneumocystis jirovecii (15)	Exclude pulmonary fungal infection
104	Negative	Aspergillus fumigatus (9)	Pulmonary fungal infection