Application of metagenomic next-generation sequencing in Diagnosing Unexplained Pulmonary Infection

doi:10.21203/rs.3.rs-3409826/v1

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Application of metagenomic next-generation sequencing in Diagnosing Unexplained Pulmonary Infection

https://doi.org/10.21203/rs.3.rs-3409826/v1

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The purpose of this study was to evaluate the value of clinical of metagenomic next-generation sequencing (mNGS) in diagnosing unexplained pulmonary Infection. A total of 99 patients with pulmonary infection were selected, and their specimens were collected and sent to traditional methods for detection, and at the same time, they were sent out for pathogen mNGS detection. The detection sensitivity, specificity and diagnostic efficiency of pathogenic microorganisms were analyzed by comparing the results of traditional detection method and mNGS sequencing. In 99 patients, pathogens were detected in 75.76% (75/99) samples by traditional assay and 95.96% (95/99) samples by mNGS. A total of 154 pathogens were detected by mNGS and 29 by traditional methods. The number and species of pathogens detected by mNGS were significantly higher than those detected by traditional culture, and the difference was statistically significant.The positive rates of bacteria, viruses, fungi and atypical pathogens detected by mNGS were 87.37%, 85.37%, 83.02% and 100%, respectively. The positive rates of bacteria, viruses, fungi and atypical pathogens detected by traditional methods were 66.32%, 19.51%, 26.42% and 14.29%, respectively.The differences were statistically significant. mNGS is superior to traditional methods in the sensitivity, specificity and diagnostic efficiency of clinical etiological detection, and has high diagnostic value of disease, thus further guiding the rational use of antibacterial drugs.

Pulmonary Infection

Metagenomic next-generation sequencing

Traditional methods

Pulmonary infection is an inflammation of lung parenchyma that is more often, but not always, caused by infections. The many causes of pneumonia include bacteria, viruses, fungi, and parasites^[1].This article will focus on the path-ogenes.Bacteria have classically been categorized into two divisions ,including "typical" and "atypical" organisms. Atyp-ical pneumonia is acquir-ed from various sources, they are even co-infection.They are difficult to culture.They presents-ubacutely and with progressive constitutional symptoms. While streptococcus pneumonia accounts for about 70% of ca-ses, the rest are caused by atypical organisms^[2]. These organisms include atypical bacterial pathogens such as Mycopl-asma pneumoniae,Legionella species and Chlamydia pneumoniae^[3].Lung infections cause an enormous global burden of disease, surpassing cancer, diabetes, HIV/AIDS, Malaria and many other diseases recognized as major global health pr-oblems^[4]. Therefore, it is particularly important to intervene in the early stage of lung infecti-on to avoid developing i-nto severe pneumonia. Microscopic smear and culture are routine methods in the diagnosis of pulmonary infection pat-hogens, but both methods are relatively insensitive and time-consuming.Histopathological diagnosis is the gold standard for invasive fungal infection.However, it takes a long time and is non-pathogen-specific.The rapid development of se-quencing technology and bioinformatics has made next-generation metagenomic sequencing (mNGS) an emerging field in clinical diagnostic development.The study of mNGS for the diagnosis of pneumonia and showed that mNGS of B-

AL provides significant value for improving the diagnosis of pneumonia and contributing to better patient care^[5]. Influ-enza virus, rhinovirus, and adenovirus frequently cause viral pneumonia, an important cause of morbidity and mortalityespecially in the extreme ages of life. During the last two decades, three outbreaks of coronavirus associated pneumon-ia,namely Severe Acute Respiratory Syndrome, Middle-East Respiratory Syndrome, and the ongoing Coronavirus Infecti-ous Disease—2019 (COVID-19) were reported^[6]. Novel techniques for pathogen detection in COVID-19 cases could becritical for versatil coinfection surveillance and appropriate antimicrobial therapy. Microbial metagenomic next generatio-n sequencing (MS) emerges as a fast-evolving technology, which allows the identification of pathogens and microbiomeinformation simultaneously within 24 h.In the SPHCC cohort,MS might be a feasible and efficient adjunctive tool for precision antimicrobial management inmechanically ventilated COVID-19 patients in ICUs^[7]. In recent years, studies ha-ve shown that the application of metagenomic next-generation sequencing plays an important role in the identification of chlamydia psittaci, mycoplasma and Legionella in severe pneumonia^[8-10].

To explore the value of mNGS in the etiological diagnosis of pulmonary infection of unknown etiology, 103 patients diagnosed with pulmonary infection in ICU from March 2019 to February 2022 who received both mNGS and conventional laboratory tests were retrospectively analyzed.The sensitivity, specificity and clinical diagnostic efficacy of mNGS detection in bacterial, fungal, viral, atypical pathogens and pneumocystis carinii lung infectious diseases were analyzed.

2.1 Patient Enrollment

Collection in March 2019 to February 2022 in patients with severe medicine diagnosis of pulmonary infection in hospital, into their baseline data, including gender, age, the main symptoms, complications, immune status, the blood oxygen saturation, inflammation index (C - reactive protein, calcitonin, the total number of white blood cells), imaging examination and laboratory traditional pathogen detection results. The following inclusion criteria were used: i)It met the diagnostic criteria of pulmonary infection. ii)The pathogen has not been identified by traditional laboratory testing methods.iii)The duration of empirical anti-infective treatment is ≥ 3 days (including the duration of anti-infective treatment in other hospitals), and the condition is not in remission; iv)The informed consent was signed and the second-generation sequencing etiology test was sent out.

2.2 Samples and Laboratory Testing

After admission, routine blood cell analysis, CRP, PCT, chest computed tomography and other examinations were performed. Sputum, blood, urine, drainage fluid and deep vein catheterization smear and culture examination were successively taken from patients according to the changes of patients conditions.At the same time, nucleic acids of 13 pathogens, antibody of respiratory pathogens, antibody and DNA of cytomegalovirus, Epstein-Barr virus and herpes simplex virus were detected, and another mNGS sample was sent to BGI testing company for pathogen detection.BAL samples were collected,of which 5 mL of the specimen was placed in a sterile sputum container, stored at -20°C, and then sent to BGI-Huada Genomics Institute. Briefly, DNA was extracted from 300 mL BAL and the sequencing library was prepared using the TIA Namp Micro DNA Kit(DP316,Tiangen Biotech,Beijing,China). Sequencing was tested using BGI SE Q-50 platform(BGI Genomics).

2.3 Definitions

Infectious pathogens were defined as those meeting either of the following conditions: culture and mNGS identified same microbe, and number of unique reads was ≥ 1 from a single species. Microbes were considered new potential pathogens if mNGS alone identified the microbe. These were based on strict clinical criteria,combined with multiple-clinician adjudication, to rigorously discriminate infection from colonization and contamination^[11].

103 patients diagnosed with pulmonary infection in ICU from March 2019 to February 2022 who received both mNGS and conventional laboratory tests were retrospectively analyzed.The sensitivity, specificity and clinical diagnostic efficacy of mNGS detection in bacterial, fungal, viral, atypical pathogens and pneumocystis carinii lung infectious diseases were analyzed.

2.4 Statistical analysis

SPSS 25.0 was used for data analysis.Counting data were represented by the number of cases and rate. Paired Chi-square test and consistency test (Mc Nemar test and Kappa test) were used for comparison between paired samples. P < 0.05 was considered statistically significant, Kappa < 0.4 was considered poor consistency, and Kappa > 0.75 was considered good consistency.

3.1 Patient Summary

A total of 99 patients with alveolar lavage fluid mNGS samples were included, including 5 patients with venous blood mNGS samples, 2 patients with pleural fluid samples, and 1 patient with sputum samples.In total, there were 66 males and 33 females with ages ranging from 32 to 95 years (mean, 60.58 years). All patients had Respiratory manifestations consistent with a pulmonary infection, Accompanied by fever, cough, expectoration, chest tightness, suffocating, dyspnea.

99 patients were routinely tested, including blood routine examination, CRP, PCTand Chest computed tomography. Blood test: blood routine white blood cell count 0.35-29.24 ×10⁹/L, with an average of 13.38×10⁹/L;100 ng/L > PCT > 0.04ng/L, ranged from 0.046 to 92 ng/L, with an average of 6.25 ng/L;CRP ranged from 2.20 to 362.40 mg/L, with an average of 127.08 mg/L.

3.2 Summary of mNGS Quality Matrices

Results of 99 patients with conventional laboratory tests and mNGS (99 BALF samples, 5 venous blood samples, 2 pleural fluid samples, and 1 sputum sample) were as follows.mNGS sequence number greater than 1 was considered as positive. Besides three samples were determined as contamination, and were treated as mNGS negative results.Traditional laboratory tests detected pathogens in 75.76% (75/99) of the samples, and MNGS detected pathogens in 95.96% (95/99) of the samples.A total of 154 pathogens were detected by mNGS, and their distribution was shown in Figure 1(A,B,C,D).A total of 29 pathogens were detected by traditional methods, and their distribution is shown in Figure 2 .

In all the results, 14 cases of mNGS detection results were basically consistent with the traditional method.The results of mNGS in 46 cases were partially consistent with the traditional method.Among these 46 cases, the mNGS detected potential pathogens that were not detected by the traditional method,including Peter acinetobacter, mannitol rolston bacteria produce indoles golden coli, solution, feed more burkholderia bacteria, Whipple disabled body, dung enterococcus, excrement enterococcus, herpes virus, parvovirus, thin ring virus, little smoke spore, rhizopus,aspergillus,candida,blastocystis,psittacosis chlamydia and mycoplasma, Ureaplasma, Actinomycetes, Anaerobic coccus, Strongyloides faecalis, etc.The results of 11 cases were inconsistent with mNGS.One case was detected by traditional method, but no pathogen was detected by mNGS.

McNemar test was used to compare the results of mNGS and traditional laboratory tests in 99 patients with pulmonary infection.Traditional laboratory and mNGS tests were performed on all 99 patients, including 73 patients who were positive for both mNGS and traditional laboratory tests, and 3 patients who were negative for both NGS and traditional clinical tests.22 patients were positive for mNGS but negative for traditional test.Test of mNGS was negative and conventional test was positive in 1 patient,as shown in Table 1. P < 0.001, the difference between the two is statistically significant, so it can be considered that the positive rate of mNGS test is higher than that of traditional laboratory test.

Table 1 Comparison of mNGS and traditional laboratory etiological test results in 99 patients

mNGS		traditional laboratory etiological test
mNGS	positive	negative	total
positive	73	22	95
negative total	1 74	3 25	4 99

According to the final results of clinical etiology, 99 patients were divided into bacterial, fungal, viral and atypical pathogen infections (pure bacterial infection and mixed infection containing bacteria were regarded as bacterial infection, and the other types of the same).Due to the small sample size of each group after classification, Fisher's exact test was used to compare the difference in detection rates between NGS and traditional methods. The results are shown in Table 2.There were 95 patients with bacterial infection, the positive rate of mNGS diagnosis was 87.37% (83/95), and the positive rate of traditional method was 66.32% (63/95), P =0.001<0.05, that is, there was a significant difference between the two detection methods, it can be considered that the diagnosis of NGS was higher than that of traditional method without significant difference.41 patients were infected with virus, and the positive rate of mNGS was 85.37% (35/41), while the positive rate of traditional method was 19.51% (8/41), P =0.000<0.05, indicating a significant difference between the two detection methods. Therefore, the diagnosis of mNGS was higher than that of traditional method.There were 53 patients with fungal infection, and the positive rate of mNGS was 83.02% (44/53), while the positive rate of traditional method was 26.42% (14/53), P value was 0.000<0.05, indicating a significant difference between the two detection methods. The diagnosis of mNGS is considered to be superior to traditional methods.Seven patients were diagnosed as atypical pathogen infection, and the positive rate of mNGS was 100% (7/7), while the positive rate of traditional method was 14.29% (1/7). The P value was 0.005<0.05, indicating a significant difference between the two detection methods.It can be concluded that mNGS is superior to traditional methods in diagnosing atypical pathogens.The distribution is shown in Figure 3 below. In addition, 72 of the total cases were diagnosed as mixed infection, and mNGS detected 50 cases, compared to only 3 cases detected by conventional methods.

Table 2 Fisher's precision test was used to compare the difference in detection raes between NGS and traditional methods.

categpory	the positive rate of mNGS	the positive rate of traditional method	P
Becterial	87.37%	66.32%	0.001
viruses	85.37%	19.51%	0.000
fungi	83.02%	26.42%	0.000
Atypical pathogens	100%	14.29%	0.005

Among the 99 patients,37 patients were immunocompromised hosts, of which 70.27% (26/37) were infected with fungi, 29.73% (11/37) were infected with Pneumocystis yersini, and 43.24% (16/37) were infected with viruses, mainly human herpesvirus, cytomegalovirus and human parvovirus.The most common bacteria were Kleiniella pneumoniae pseudomonas aeruginosa and Acinetobacter baumannii, and the rest were Escherichia coli, Enterococcus faecium and Legionella.

3.3 Analysis of the cause of the contradiction between M NGS and traditional etiological detection in this study

Among the 99 patients, The mNGS sequencing results of 11 patients were inconsistent with the results of traditional laboratory tests. We combined the clinical symptoms, relevant laboratory test results and treatment effect of the patients. The traditional laboratory test results were considered as contamination in 3 cases, mNGS results were considered as contamination in 3 cases, and the remaining 5 cases were considered as co-infection.

3.4 Treatment and outcome

All patients were given empirical anti-infective treatment on admission according to their medical history, clinical manifestations, relevant imaging and laboratory test results, etc., and the treatment plan was adjusted in time after the pathogen type was identified.

12 cases were diagnosed with pneumocystis pneumoniae and treated with compound sulfamethoxazole.3 patients were diagnosed as Legionella pneumonia and were treated with azithromycin and moxifloxacin.17 patients were diagnosed with aspergillus infection and treated with carbofengin, voriconazole or amphotericin B.Klebsiella pneumoniae,Pseudomonas aeruginosa, acinetobacter baumannii and other infections were treated with sensitive antibiotics according to drug sensitivity results.Acyclovir was used to treat human herpes virus, ringlet virus, rhinovirus and other infections. After personalized anti-infection treatment for pathogens and supportive treatment for complications and complications, 45 patients improved and were discharged from hospital, 23 patients were discharged from hospital automatically, and 31 patients died and were discharged from hospital due to disease progression and multiple organ failure.

Pulmonary infection is caused by common pathogens，inhalation injury, tracheostomy，and patients often have different clinical manifestations.Patients in the intensive care unit (ICU) are critically ill and frequently have multiple diseases，beccause of poor resistance and decreased body immunity.These lead to increased risk of patient morbidity, length of hospital stay and treatment costs.In recent years, with the development of Metagenomics next-generation sequencing, they makes lung infection pathogen early detection, for the disease diagnosis and treatment to provide a great help^[12].

In our study, mNGS exhibited better performance than conventional test for detecting bacte-ria (p<0.05), this study showed that the sensitivity of mNGS in pulmonary infection pathogen detection was much higher than that of the co-nventional test(p<0.01). This was inconsistent wi-th previous findings.According to Xie et al, the sensitivity of mNGS is better than that of cul-ture for recognizing bacteria, while the specificity of the conventional test was better than m-

NGS^[13]. In a prospective study with 138 patients from a single center,they performed metagen-omic mNGS of bronch-oalveolar lavage fluid for pathogen identification in pneumonia. mNGS identified more bacteria than standard methods

^[14]. With the increase of the immunosuppressed population, community-acquired pneumonia in immunosuppressed patie-nts has become an important part of the intensive care unit. Without rapid diagnosis, the disease can rapidly progress to respiratory failure, sepsis, and multiorgan failure^[15]. Sixty-nine immunocompromised patients with suspected pneumo-nia were carried out with conventional test and mNGS of BALF,and theoverall detection rate of mNGS was higher th-an that of conventional detection^[16].

In the identification analysis of virus infection by mNGS and conventional test,this study also showed that the se-nsitivity of mNGS in pulmonary infection pathogen detection was muchhigher than that of the conventional test (p<

0.05). Tetected viruses were mainly HSV,troque ten-ovirus,human parvovirus B19, rhinovirus and influenza B virus. Al-though the diagnostic rate ofmNGS is higher than that of conventional test.The diagnosis of viral infection mainly reli-es onrespiratory virus screening, CMV-DNA, etc. mNGS has certain dia-gnostic limitations. Accordi-ng to Wanghui Shi, et al., In the identification and analysis of BALF and sputum samples by mNGS, the coincidence rate of virus detec-tion was 71.88%. they concluded that there was no- significant difference in mNGS results between BALF and sputumsamples^[17]. These are notconsistent with our findings.Though the mNGS test can detect a variety of viruses, this does not mean that mNGS is more efficient than PCR.Reporting identified viruses also requires cau-tion, all viruses are not pathogenic and some of them, such as herpesviruses, colonize patients for long periods of time and/or integrate their genetic material into host cells^[18].

In recent years, metagenomic next generation sequencing (mNGS) has been increasingly widely used in clinical dia-gnosis, which has remarkable application value in the diagnosis of fungal infections.Due to the widespread use of bro-adspectrum antibiotics, hormones, antitumor drugs, immunosuppressants and the extensive development of various invas-ive procedures, the incidence of fungal infections has increased.Fungal infections, such as pulmonary aspergillosis, inva-sive candidiasis, cryptococcosis, or histoplasmosis, are associated with high mortality.In the identification analysis of fu-ngi infection by mNGS and conventional test,our study also showed that the sensitivity of mNGS in pulmonary infect-ion pathogen detection was much higher than that of the conventional test(p<0.05).Zheng et al. summarized the diagno-stic value of mNGS for lower respiratory tract fungal infections and the impact of different sampling methods on the detection efficiency of mNGS. The mNGS can detect fungal pathogens that are difficult to diagnose by traditional det-ection methods.However, mNGS has no obvious diagnostic advantage in cryptococcal detection. Qian et al.described th-e diagnosis of lung infections by mNGS and reported the performance of mNGS against various Candida species. Thesensitivity of mNGS for the detection of pathogens and fungi was higher than that of traditional cultures^[19].Lung biopsyand BALF samples were obtained from 106 patients with suspected pulmonary fungal i-nfection by bronchoscopy, the sensitivity of mNGS(lung biopsy, BALF) in the diagnosis of pu-lmonary fungal infection was significantly higher than that of routine examination^[20].

In recent years, the incidence and mortality of atypical pathogen pneumonia are increasing year by year. The traditional pathogen detection level is limited.The application of mNGS is helpful for the early diagnosis of the disease.In

the identi-fication analysis of atypical pathoge-ns infection by mNGS and conventional test,our study also showed thatthe sensitivity of mN-GS in pulmonary infection pathogen detection was much higher than that of the conventional test(p<0.05).In a Case Report of Community-Acquired Legion-ella gormanii Pneumonia, routine culture of bronchoalveolar lavage fluid and urinary antigen test for Legionella were negative. However, Metagenomic nextgeneration sequencing (mNGS) revealed a large number of DNA a-nd RNA sequences of Legionella gomagei in bronchoalveolar lavage fluid,but not in blood sa-mples^[9]. The incidence of actinomycosis was once thought to be low, but in recent years the inci-dence of actinomycosis has increased with the increase in the incidence of tumors, organ transplantation, and the use of immunosuppressive agents.In addition, this anaerobic bacterium is difficult to identify using conventional methods b-ecause it requires very specific growth conditions.Fortunately, mNGS has the advantage of recognizing an unusually a-bundant actinomycetes.According to Yang et al, mNGS found a large number of actinomycetes in the BALF of the t-wo patients, with unique reads greater than 500, and actinomycetes accounted for more than 30% of all organisms de-tected in the two samples^[18].In the diagnosis of pneumocystis pneumo-nia in non-HIV immunosuppressed patients, the sensitivity and specificity of BALF mNGS in the diagnosis of Pneumocystis pneumonia were 97.40% and 85.12%, respectively^[21].

In previous studies on Pneumocystis gireni pneumonia, it was found that the detection time of mNGS (24 h) for Plasmodium gireni was significantly faster than that of GMS (5-7 days), and the test results were consistent with the

traditional test results.The mNGS method had absolute advantages^[23]. A retrospective study showed that the sensitivity of mNGS in the diagnosis of PJP was 100%, significantly higher than that of GMS staining (25.0%) and plasma β-(1,3) -d glucan (67.4%).The specificity of mNGS (96.3%) was significantly higher than that of plasma β-(1,3) -d glucan (81.4%)^[24].According to Ju et al，Their findings suggest that mNGS has considerable clinical value for the diagnosis ofatypical pathogens. Other pathogens such as Legionella pneumoniae, Mycoplasma pneumoniae, and Strongylus faecalis were detected by mNGS in BALF, which is consistent with our findings^[22].

In recent years, mNGS have been widely used in infectious diseases,The specimen types include blood, cerebros-pinal fluid, pleural fluid and lung tissue, etc., thus providing evidence for antibiotic treatment^[25-27].In a Case Report ofDisseminated Mucormycosis, In recent years,mNGS has been widely used in infectious diseases. The types of specime-ns include blood, cerebrospinal fluid, pleural effusion and lung tissue^[28]. Antimicrobial resistance (AMR) is rising at analarming rate and complicates the management of infectious diseases, including lower respiratory tract infections (LRTI).In contrast to clinical antimicrobial susceptibility tests, the performance of respiratory mNGS in predicting antibiotic re-sistance varies by pathogen, antimicrobialagent, and nucleic acid type sequencing. mNGS can be used to detect and m-onitor LRTI pathogens^[29].

This study has some limitations.Due to the limitations of mNGS technology, its application is still controversial. First of all, the study sample is small, which may affect the accuracy of mNGS performance evaluation.Secondly, the accuracy of mNGS is affected by many factors, such as sample processing, bioinformatics analysis, and interpretation of sequencing results. For small sample sizes, we still need more data to reflect the pathogen spectrum in patients with pulmonary infections.With the reduction of sequencing cost and the development of technology, the cost of mNGS has been reduced to an acceptable level, which will be more and more applied in clinical research and treatment.

Ethics approval and consent to participate

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Hospital of Shandong Uniersity. Written informed consent was obtained from individual or guardian participants.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in,or the review of,the manuscript entitled.

Funding

Not applicable.

Authors' contributions

Jinpeng Yu:Conceptualization, Methodology, Software, Investigation,Data Curation,Formal Analysis, Software, Software,Software, Writing - Original Draf.

Wei Gao : Conceptualization, Resources, Supervision, Visualization,Writing - Review & Editing.

Authors' information

Yu Jinpeng,Department of Internal Medicine,Maternal and child health hospital of Lin’yi City,Lin'yi City,276017, Shandong Province, China (e-mail: [email protected]).

Wei Gao,The Second Hospital of Shandong Uniersity, Ji'nan City,250100, Shandong Province, China (e-mail: [email protected]).

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Application of metagenomic next-generation sequencing in Diagnosing Unexplained Pulmonary Infection

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Abstract

1. Introduction

2. Materials and Methods

2.1 Patient Enrollment

2.2 Samples and Laboratory Testing

2.3 Definitions

2.4 Statistical analysis

3. Results

4. Discussion

Declarations

References

Additional Declarations

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