Sepsis, a major public health problem, is a syndrome physiological, pathological, and biochemical abnormality caused by infection (Singer et al., 2016). About 1.94–31.5 million deaths occur annually worldwide, of which about 20% (5.3 million deaths) are sepsis-related (Fleischmann et al., 2016; Maraki et al., 2016; Singer et al., 2016; Rudd et al., 2020). Sepsis is also the leading cause of admission to an intensive care unit (ICU) and death (Abe et al., 2018). The common infection sites include lung, abdomen, blood, urinary tracts, and central nervous system. Severe sepsis requires that the infection site, microbial species, and empirical anti-infection treatment are identified first (Rhodes et al., 2017; Cecconi et al., 2018; Niederman et al., 2021). In sepsis management strategies, early recognition and standardized management such as fluid resuscitation and hormone use are proposed, but active antibiotic use remains the cornerstone of successful sepsis treatment (Rhodes et al., 2017; Cendejas-Bueno et al., 2019; Niederman et al., 2021). Pathogen identification and beginning or adjusting the antibiotic therapy as soon as possible are essential. A traditional bacterial culture takes 3–5 days, and specific pathogenic bacteria are difficult to culture, take a longer time, have a low positive rate, difficult to diagnose, and have ineffective and delayed empirical treatment.

Metagenomic next-generation sequencing (mNGS) is a new method that combines high-throughput sequencing with bioinformatics analysis; its advantages include shorter detection time of microorganisms, accurate detection of multiple pathogens such as bacteria, fungi, viruses, and parasites at one time through DNA or RNA gene sequencing of clinical samples (Church et al., 2020; Vandenberg et al., 2020; Evans et al., 2021). This method provides a clear etiological basis for severely infected patients, and allows more targeted medication. However, as a new detection method, it is expensive with unclear clinical characteristics and influencing factors in ICU and there are few relevant studies. Thus, this study analyzed and discussed the clinical characteristics and influencing factors of mNGS in ICU.

Severe infections in ICU patients progress and change rapidly. Sepsis guidelines (Cecconi et al., 2018; Dugar et al., 2020) recommend identifying the infection site first, followed by collecting the body-fluid specimens for routine culture, identifying pathogens, and finally, selecting the targeted antimicrobial drugs. The correct use of antimicrobial agents in the early stages is critical for the prognosis of patients with sepsis or septic shock. However, the positive rate of a traditional blood culture is about 20% lower and about 3–5 days longer (Greninger and Naccache, 2019). For particular bacteria cultures, Mycobacterium tuberculosis needs a longer culture time, and the positive rate of pathogens such as Nocardia, Cryptococcus, and Brucella is lower, while viruses fail to culture (Mancini et al., 2010; Siwakoti et al., 2018; Zhang H. C., et al., 2019; Li et al., 2021), thus causing a delay in the treatment. The mNGS can be obtained within 24 hours, has unique advantages for the traditional bacterial culture, and helps screen the pathogenic bacteria, thereby guiding the clinical application better. We discussed the clinical characteristics and value of its function as a new pathogen test in patients with severe sepsis in ICU.

A total of 124 patients with severe sepsis were included in this study, most of whom received vasoactive drugs, had a high APACHE II score, undergone tracheotomy and transferred from sub-hospitals. According to the pathogens found: G-bacillus accounted for the highest proportion; the top two pathogens were Klebsiella pneumoniae, Acinetobacter baumannii, which was consistent with the current epidemiology of hospital-related infections (Kaye and Pogue, 2015; Dugar et al., 2020; Geng et al., 2021). Additionally, one case of Pneumocystis and one tuberculosis pathogens were found. Pneumocystis jirovecii was found in mNGS of a patient with a malignant hematologic tumor complicated with septic shock. Related studies have also shown that although pneumocystis is almost exclusively present in the human lung, pneumocystis fragments can enter the peripheral blood through the site of respiratory infection, especially under immunosuppression (Zhang Y., et al., 2019; Chen et al., 2020; Zhang et al., 2021). It suggests that blood mNGS can help diagnose Pneumocystis jirovecii, which is difficult to diagnosed. In another case of immune-related disease, fragments of bacterium tuberculosis was found in the blood and led to septic shock. This was very challenging for the clinician to determine and distinguish (Mishra et al., 2019; Sun et al., 2021). The mNGS fungi detection rate was significantly higher than the blood culture results with significant statistical differences, and virus detection was impossible in the blood culture. This is consistent with the views of Zheng et al. (Zheng et al., 2022). Highlighting the clinical advantages of NGS in finding particular fastidious pathogenic bacteria that can guide the clinical treatment better (Kruppa et al., 2018; Duan et al., 2021b; Govender et al., 2021; Tsang et al., 2021; Zhan et al., 2022).

This study showed that the mNGS positive rate was higher, and the mNGS pathogen coverage accounted for 54.17% (13/24) of the total number of positive blood culture results, and two other patients showed positive results only in blood cultures. This indicates that mNGS may miss pathogens, be inconsistent with blood culture, have false negative results, and need for higher sequencing depth detection (Duan et al., 2021a; Gu W., et al., 2021). Natoli et al. (2022) also agreed that any contamination with the blood culture between sample processing and data analysis could bias the final results seriously. Boers et al. (Boers et al., 2019) also showed that the above reasons lead to the discovery of non-existent bacterial genera, false correlations between microbes and their hosts, and the inability to detect true correlations. However,The advantages of mNGS detection were not affected by history of antimicrobial exposure. In 2019, Blauwkamp et al. (Blauwkamp et al., 2019) found that microbial cell-free DNA (mcfDNA) sequencing test performed much better than blood culture in analyzing specimens from subjects who had received antimicrobial therapy within two weeks preceding presentation. The reason is that microbial sequencing methods diagnose possible infections by capturing and identifying this highly fragmented mcfDNA in the circulating system, which are lysed fragments of bacteria and conventional blood culture failed to detect (Han et al., 2020).

Our study reported that mNGS showed a higher positive rate in patients having immune-related diseases with sepsis, and a statistically significant higher detection rate of single pathogen infection compared to the no immune-related diseases in patients with sepsis. The earlier studys (Geng et al., 2021; Yan et al., 2021) showed that low immunity was associated with multiple infections. Niles et al., (Niles et al., 2022) in a retrospective study of 169 participants, showed that immune-related patients were more likely to obtain multiple microbial results from mNGS than patients with normal immune function.

Subsequently, we analyzed the influence of sepsis patients with negative culture and mNGS results and positive results of mNGS or blood culture bacteriology. Univariate analysis and multivariate analysis suggesting that sepsis patients with high APACHE II score, immune-related diseases, hypertension had lower double-negative results. This means the more severe the disease, the easier it is to obtain positive blood cultures or mNGS results. There were few reports on the clinical influencing factors of mNGS results. As far as we know, only one study shows that age is a significant influencing factor in the multi-factor logic analysis of positive mNGS results (Duan et al., 2021a).

We included mNGS reads in univariate and multivariate analyses of its impact on the prognosis of patients with sepsis. Our research found that microbial sequence number and pathogen detection (mNGS) type had no direct relationship to the prognosis of sepsis. And age, female, myoglobin and virus-positive results were independent risk factors for sepsis. This suggests that mNGS reads represent the presence of certain bacterial infections, and cannot be associated with disease prognosis. Ong et al. (Ong et al., 2017) showed in a prospective study center that 68% of the septic shock patients activated after viral infection without prior immune deficiency were independently associated risk factors for sepsis mortality. Age, female, and myoglobin are more analyzed in the influencing factors of sepsis (Yang et al., 2019; Gu B., et al., 2021).

This study showed no statistical difference in the overall prognosis of sepsis according to mNGS adjustment regimen, and this may be related to the source of patients, severe septic shock, and small sample size. However, there is no doubt that mNGS can be used to quickly, and a high positive rate to obtain pathogens included fastidious bacteria (Huang et al., 2019). The results of Geng et al. (Geng et al., 2021) were in agreement with the above-mentioned results. mNGS has obvious advantages as it can quickly, efficiently, and accurately obtain all nucleic acid information in test samples, analyze pathogens, guide clinical diagnosis and treatment, and find viruses, fungi, parasites, rare pathogens, and even unknown pathogens (Consensus Group Of Experts On Application Of Metagenomic Next Generation Sequencing In The Pathogen Diagnosis In Clinical et al., 2020; Han, 2022). Zhou et al. (Zhou et al., 2021) described the use of mNGS for pneumonia pathogen identification in a large-scale multi-center prospective study of 159 patients, which resulted in 59 patients (37.1%) changing treatment regimens, including 40 patients (25.2%) downgrading antibiotic use. There are few studies on the application of mNGS in ICU patients, so it is necessary to expand the sample size and further explore the effect of adjusting antibacterial application according to the mNGS results on the prognosis of ICU patients with sepsis.

This study has some limitations. First of all, the origin of the patients (transfered from subordinate hospitals and in a critical condition) may cause negative mNGS and blood culture results. In addition, the antibiotic exposure history has a greater impact on blood culture, leading to an increase in the negative rate of blood culture. Secondly, our sample size is small. Therefore, further large-sample studies are needed to explore the application of mNGS in sepsis. Thirdly, the study does not represent the application characteristics of mNGS in other infections, fever and pathogenic bacteria in difficult cases, and in critically ill patients in other regions.

In conclusion, mNGS can quickly obtain pathogenic bacteria (including fastidious bacteria), have a higher positive rate. The mNGS positive rate was higher than that of the blood culture, but there are still some negative results. Patients with high APACHE II score, immune-related diseases, hypertension had lower double-negative results. The sequence number of microorganisms and pathogen detection (mNGS) type such as negative,infection with a single pathogen and mixed infection were not directly related to the disease prognosis. There was no statistical difference in prognosis of sepsis according to mNGS adjustment regimen. However, mNGS is very important for the acquisition of pathogenic bacteria in severe patients, and we would carry out a larger sample study to explore the impact of mNGS on the outcome of sepsis bloodstream infection.

The data presented in the study are deposited in the ERP repository, accession number PRJEB53676.

The studies involving human participants were reviewed and approved by The study was approved by the Scientific Research and Clinical Trials Ethics Committee of the First Affiliated Hospital of Zhengzhou University (code 2021-KY-0600-002). The patients/participants provided their written informed consent to participate in this study.

LMS and SGZ were responsible for managing the patient. ZYY and FY collected data. ZHW, HQL, and TWS revised the article. All authors have read and approved the final manuscript and contributed substantially to the work presented in this article.

This study was supported by the 51282 Project Leaders of Scientific and Technological Innovative Talents from Health and Family Planning Commission in Henan Province (2016-32), and Science and Technology people-benefit project of Zhengzhou (2019KJHM0001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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