Clinical Application and Evaluation of Metagenomic Next-Generation Sequencing for Lower Respiratory Tract Infections and Human Tumor Screening

Figures & data

Table 1 Comparison of General Data Between LRTIs and Non-LRTIs

	LRTIs (n = 149)	Non-LRTIs (n = 39)	P
Age, mean (range), years	60 (51, 69)	59 (50, 69)	0.785
Sex, female, n (%)	32.2	33.3	1.000
Underlying disease, n (%)	83.9	59.0	0.001
Antibiotic use before mNGS, n (%)	75.2	53.8	0.011
White blood cell count, 10^9/L	7.50 (5.55, 11.00)	8.10 (5.70, 10.20)	0.772
White blood cell count, >9.5×10^9/L	31.5	41.0	0.341
Neutrophil count, 10^9/L	5.50 (3.55, 8.80)	5.50 (3.60, 8.70)	0.917
Neutrophil count, >6.3×10^9/L	40.9	41.0	1.000
C-reactive protein, mg/L	36.08 (3.69, 106.54)	4.29 (0.50, 29.67)	< 0.001
C-reactive protein, >10 mg/L	66.9	43.6	0.010
Procalcitonin, ng/mL	0.09 (0.04, 0.27)	0.05 (0.03, 0.09)	0.003
Procalcitonin, >0.05 ng/mL	65.7	50.0	0.087

Notes: Data are presented as n (%) or mean (range). Significance was determined by Chi-square test or non-parametric rank sum test.

Abbreviations: LRTIs, lower respiratory tract infections; mNGS, metagenomic next-generation sequencing.

Figure 1 Flow diagram showing patient enrollment. ^aUncertain pathogen, it is unclear whether the detected pathogen is pathogenic or colonizing bacteria; ^bRespiratory tract specimens, including sputum, alveolar lavage fluid, and lung biopsy tissue.

Abbreviations: LRTIs, lower respiratory tract infections; LRTDs, lower respiratory tract diseases.

Table 2 Comparison of the Pathogen Detection Techniques

	mNGS (n = 188)	Traditional Culture (n = 180)	CMT (n = 186)	P
Sensitivity (%)	65.8	21.8	38.1	< 0.001
Specificity (%)	69.2	94.7	43.6	< 0.001
Accuracy (%)	66.5	37.2	39.2	< 0.001
PPV (%)	89.1	93.9	71.8	0.001
NPV (%)	34.6	24.5	15.7	0.012

Notes: The diagnostic sensitivity and accuracy of the mNGS group were higher than those of the traditional culture and CMT groups (P < 0.001; P < 0.001; P < 0.001; P < 0.001), and the specificity was higher than that of the CMT group (P = 0.039) but lower than that of the traditional culture group (P = 0.006). Data were presented as n (%). Significance was determined using the Chi-squared test or Fisher’s exact probability method.

Abbreviations: CMT, comprehensive microbial test; mNGS, metagenomic next-generation sequencing; NPV, negative predictive value; PPV, positive predictive value.

Table 3 Comparison of Detection Rates of Different Pathogen Types Between mNGS and CMT

	mNGS (n, %)	CMT (n, %)	P
Single bacteria (n = 59)	56 (94.9)	33 (55.9)	< 0.001
MTB/NTM ^a (n = 29)	29 (100)	13 (44.8)	< 0.001
MTB ^a (n = 21)	21 (100)	10 (47.6)	< 0.001
Atypical pathogen ^b (n = 10)	10 (100)	3 (30.0)	0.003
Chlamydia psittaci (n = 8)	8 (100)	3 (37.5)	0.007
Single fungus (n = 24)	24 (100)	14 (58.3)	< 0.001
P. jirovecii ^a (n = 18)	18 (100)	4 (22.2)	0.001
C. neoformans ^a (n = 7)	6 (85.7)	7 (100)	1.000
Mixed pathogens (n = 20)	18 (90.0)	5 (25.0)	< 0.001

Notes: ^aThe number of pathogens includes single infections and mixed infections. ^bAtypical pathogens include Chlamydia psittaci, Legionella hackeliae, and Chlamydia abortus. Data were presented as n (%). Significance was determined using the Chi-squared test or Fisher’s exact probability method.

Abbreviations: mNGS, metagenomic next-generation sequencing; CMT, comprehensive microbial test; MTB, Mycobacterium tuberculosis complex; NTM, non-tuberculous Mycobacteria; P. jirovecii, Pneumocystis jirovecii; C. neoformans, Cryptococcus neoformans.

Table 4 Clinical Data of 15 Cases of Malignant Tumor with CNVs and Pathogens Detected by mNGS

Case Number	Age/Sex	Sample	Imaging Results	Tumor History	Primary diagnosis	Definitive diagnosis	Diagnostic method	CNV detection`	Pathogens detected by mNGS
[1]	62/M	PB	Multiple masses with cavities	Left RUC after surgery	Lung abscess*	Lung metastasis	Pathology	Positive	Positive^¶a
[2]	83/M	PB	Large mass with cavity in RUL	None	Lung abscess*	PLA	Pathology	Positive	Negative
[3]	51/M	PB	Multiple lung nodules, opacities with cavities	Liver cancer with lung metastasis	MLC with lung abscess	MLC with lung abscess	Clinical diagnosis	Positive	Negative
[4]	66/M	PB	Bilateral lung opacities, multiple masses in the liver	None	Liver abscess with pneumonia*	PHAA with pneumonia	Pathology and Clinical diagnosis	Positive	Negative
[5]	62/M	PB	A mass in LLL with bilateral pleural effusion	Systemic metastasis of left maxillary of ACC after surgery	Pneumonia secondary to MLC	Pneumonia secondary to MLC	Clinical diagnosis	Positive	Negative
[6]	76/M	PB	GGOs in both lungs	Systemic metastasis of right maxillary of ACC	Pneumonia	Radiation pneumonitis	Clinical diagnosis	Positive	Positive^¶b
[7]	72/M	LT	Cystic lesion in RLL	None	Fungal pneumonia*	PLA	Pathology	Positive	Negative
[8]	75/M	LT	Multiple nodules in RLL	None	Suspected lung cancer	PLA	Pathology	Positive	Negative
[9]	86/M	LT	Multiple nodules in both lungs	PA with lung metastasis	Secondary fungal pneumonia*	Lung metastasis	Pathology	Positive	Negative
[10]	64/F	LT	Multiple nodules in LLL	None	Fungal pneumonia*	PLA	Pathology	Positive	Negative
[11]	75/M	BALF	Multiple lung bulla, consolidation	None	Pneumonia*	GA with LM	Pathology	Positive	Positive^¶c
[12]	65/M	BALF	Consolidation in RLL	None	Pneumonia*	PLA	Pathology	Positive	Negative
[13]	64/F	BALF	Multiple nodules, masses and consolidations	Lung cancer with systemic metastasis	Pneumonia secondary to lung cancer	Pneumonia secondary to lung cancer	Pathology, bacteriology	Positive	Positive^¶d
[14]	68/F	BALF	Multiple nodules in both lungs and consolidations in LLL	None	Pneumonia*	PLA	Pathology	Positive	Positive^¶e
[15]	29/F	BALF	A nodule with cavity in LUL and multiple GGOs in left lung	None	Pneumonia*	PLA	Pathology	Positive	Positive^¶f

Notes: *These cases were initially misdiagnosed as non-neoplastic diseases, with a misdiagnosis rate of 66.7% (10/15). ^¶Non-pathogenic bacteria according to comprehensive clinical adjudications. ^aCytomegalovirus; ^bEnterococcus faecalis, Epstein-Barr virus; ^cStaphylococcus haemolyticus, Candida albicans, Cryptococcus neoformans Grubii variant, Epstein-Barr virus; ^dPneumocystis jirovecii; ^eStenotrophomonas maltophilia.

Abbreviations: ACC, adenoid cystic carcinoma; BALF, bronchoalveolar lavage fluid; CNV, copy number variation; GA, Gastric adenocarcinoma; GGOs, ground glass opacities; LLL, left lower lung; LNB, lymph node biopsy; LT, lung tissue; LUL, left upper lung; mNGS, metagenomic next-generation sequencing; MLC, metastatic lung cancer; PA, prostate adenocarcinoma; PB, peripheral blood; PHSA, primary hepatic sarcomatoid carcinoma; PLA, primary lung adenocarcinoma; RLL, right lower lung; RUC, renal urothelial carcinoma; RUL, right upper lung.

Figure 2 (A) Chest computed tomography scan showing multiple pulmonary bullae, multiple patchy and large flake consolidation in both lungs, with predominance in the lower lobes. (B) Electronic gastroscopy revealing an irregular mass in the fundus of the stomach involving the upper body of the stomach. (C) Stomach biopsy revealing poorly differentiated adenocarcinoma. (D) Biopsy of the left lower lobe of the lung revealing invasive adenocarcinoma, originating from the stomach.

Figure 3 Chromosome detection revealing genome instability due to CNV, indicating a highly suspected tumor.