CN110964778B - Use of enterobacteriaceae as ischemic cerebral apoplexy biomarker - Google Patents

Abstract

The invention relates to the field of biomarkers, in particular to application of enterobacteriaceae as an ischemic cerebral apoplexy biomarker. The inventor discovers that enterobacteriaceae can be used as a novel biomarker for evaluating the risk of the apoplexy, diagnosing the variety of the apoplexy, evaluating the prognosis of the apoplexy and the like, and has great significance in early recovery ending and long-term function ending, so that the enterobacteriaceae can be developed into a novel biomarker in accurate medicine.

Description

Enterobacteriaceae as biomarkers for ischemic stroke

Technical field

The present invention relates to the field of biomarkers, and in particular to the use of Enterobacteriaceae as biomarkers for ischemic stroke.

Background technique

Identifying the risk of adverse stroke outcomes is important for clinical management, but not all patients can achieve satisfactory recovery with previous standardized treatments. For doctors and patients, identifying patients with a higher risk of adverse EI is very important for doctors to adjust treatment plans or develop new treatments.

Contents of the invention

In view of the above shortcomings of the prior art, the purpose of the present invention is to provide the use of Enterobacteriaceae as a biomarker for ischemic stroke to solve the problems in the prior art.

In order to achieve the above objects and other related objects, one aspect of the present invention provides the use of substances for detecting Enterobacteriaceae in preparing a kit for evaluating the therapeutic effect of ischemic stroke and/or judging the deficiency. Prognosis of hemorrhagic stroke.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting Enterobacteriaceae in fecal samples.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting Enterobacteriaceae in the intestine.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting the enrichment degree of Enterobacteriaceae.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting bacterial 16S rRNA.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting Enterobacteriaceae in the large intestine.

In some embodiments of the present invention, the substance for detecting Enterobacteriaceae is a substance for detecting the 16S rRNAV4 region of bacteria.

In some embodiments of the present invention, the kit is used to evaluate the therapeutic effect of ischemic stroke and/or to determine the prognosis of ischemic stroke based on the enrichment degree of Enterobacteriaceae in the sample.

In some embodiments of the invention, the sample is a fecal sample.

In some embodiments of the present invention, the ischemic stroke is NIHSS≥4.

In some embodiments of the invention, the ischemic stroke is selected from complete stroke.

In some embodiments of the invention, the ischemic stroke is in the acute phase.

In some embodiments of the present invention, the evaluation of the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke specifically refers to evaluating the early recovery outcomes and/or long-term functional outcomes of patients with ischemic stroke. .

Another aspect of the present invention provides the use of a substance for detecting Enterobacteriaceae in the preparation of a kit for assessing whether a subject is susceptible to ischemic stroke with a poor prognosis.

Another aspect of the present invention provides the use of a substance for detecting Enterobacteriaceae in the preparation of a kit for diagnosing ischemic stroke.

Description of drawings

Figure 1 shows the comparison of the bacterial flora of EI and poor EI subgroups in the modeling cohort of the present invention. (A) PCoA plot shows significant differences in bacterial communities between the EI group and the poor EI group. (B) Mean relative abundance of major bacterial taxa across families, phyla, and genera in patients with EI and poor EI. (C) LEfSe identifies the taxa that differ most between the two groups. Only the Enterobacteriaceae and Enterobacteriaceae taxa have effective LDA thresholds >4. (D) Box plot showing the abundance of Enterobacteriaceae in the poor EI group compared with the EI group. PC, principal coordinate analysis (PCoA). EI, patients with EI (n=15); poor EI, patients without EI (n=21).

Figure 2 shows a schematic diagram of the present invention comparing the microbial community of EI and the adverse EI subgroup in the validation cohort, wherein (A) the PCoA plot shows that there is no significant difference in the bacterial community between the EI group and the adverse EI group in the validation cohort ; (B) Mean relative abundance of major bacterial taxa at the department level in EI and poor EI patients; (C) LEfSe identifies taxa that differ most between the two groups. Only Gammaproteobacteria, Enterobacteriaceae, Enterobacteriaceae, and Proteobacteria taxa meet the LDA valid threshold >4; (D) Box plot showing the abundance of Enterobacteriaceae in the poor EI group compared with the EI group . PC, principal coordinate analysis (PCoA). EI, patients with EI (n=37); poor EI, patients without EI (n=51).

Figure 3 shows a schematic diagram of the predictive performance of adverse EI outcomes of the present invention, wherein (A) the predictive performance of the model (Enterobacteriaceae + fasting blood glucose) on adverse EI outcomes in the modeling cohort is 78.8% (area under the curve [AUC ] estimate, 78.8%), while Enterobacteriaceae had similar predictive performance for adverse EI (AUC estimate, 76.9%); (B) In the validation cohort, the area under the curve for Enterobacteriaceae for adverse EI outcome was 70.2%. , while the predictive model of Enterobacteriaceae plus fasting blood glucose was 73.3%.

Figure 4 shows a schematic diagram of the present invention's model for predicting 90-day best outcomes and good outcomes, wherein (A) adding adverse EI outcomes to the models for predicting 90-day best outcomes and good outcomes significantly increases their predictive performance; (A) B) The addition of adverse EI outcomes increased the predictive performance of optimal 90-day outcomes and favorable outcomes in the entire patient cohort (combined modeling and validation cohorts) (C) The addition of Enterobacteriaceae also increased the predictive performance of optimal 90-day outcomes and favorable outcomes in the entire patient cohort (combined modeling and validation cohort) .

Detailed ways

The inventor of the present invention discovered through extensive research that Enterobacteriaceae is closely related to the diagnosis and treatment effect of ischemic stroke and/or the prognosis of ischemic stroke, and based on this, the present invention was completed.

A first aspect of the present invention provides the use of substances for detecting Enterobacteriaceae in preparing a kit for evaluating the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke. The ischemic stroke is a general term for brain tissue necrosis caused by occlusion of the blood supply arteries to the brain and insufficient blood supply. The cause of vascular occlusion can be cerebral embolism, cerebral thrombosis, etc.

Stroke mainly includes hemorrhagic stroke and ischemic stroke. Among them, ischemic stroke accounts for 60-70% of strokes. Ischemic stroke mainly includes: transient ischemic attack (TIA); complete stroke (CS). In a specific embodiment of the present invention, complete stroke is preferably targeted. In another specific embodiment of the present invention, the ischemic stroke is a patient with moderate to severe stroke (NIHSS score ≥ 4, and the scoring method of NIHSS (National Institute of Health stroke scale) can be found in Williams LS, Yilmaz EY, Lopez-Yunez AM. Retrospective Assessment of Initial Stroke Severity with The NIH StrokeScale. Stroke. 2000;31:858–862). In another specific embodiment of the present invention, the ischemic stroke is in the acute phase, and the acute phase usually refers to within 7 days of onset.

In the present invention, the substance used for detecting Enterobacteriaceae can be various detection products suitable for detecting Enterobacteriaceae in the field, for example, it can be a PCR kit, a FISH kit, etc., and the target of the detection product is It can be a stool sample, which can better reflect the large intestine intestinal flora, or it can be a 16S rRNA detection of bacteria, preferably a 16S rRNA V4 region of bacteria, so as to obtain information about Enterobacteriaceae in the sample. In a specific embodiment of the present invention, the substance for detecting Enterobacteriaceae may be a substance for detecting Enterobacteriaceae in fecal samples. In another specific embodiment of the present invention, the substance for detecting Enterobacteriaceae can be a substance for detecting Enterobacteriaceae in the intestine, preferably the large intestine. The sample to be detected usually reflects the intestinal tract of the patient. Enterobacteriaceae in the tract. In another specific embodiment of the present invention, the substance for detecting Enterobacteriaceae may be a substance for detecting the enrichment degree of Enterobacteriaceae, and the enrichment degree may be in a certain amount of sample (for example, unit Volume, unit mass, etc.) Enterobacteriaceae content, percentage in the bacterial flora, etc. In another specific embodiment of the present invention, the method of using the substance and/or kit for detecting Enterobacteriaceae may include the following steps: obtaining the total DNA of intestinal flora in the sample, and amplifying the bacterial 16SrRNA V4 region gene Based on the characteristic tag sequence, the enrichment information of Enterobacteriaceae in the sample can be obtained based on the amplification results. For the classification of bacterial types, the OTU (Operational Taxonomic Units) method can be used.

In the present invention, the kit is used to evaluate the therapeutic effect of ischemic stroke and/or to determine the prognosis of ischemic stroke based on the enrichment degree of Enterobacteriaceae in the sample. Preferably, the sample targeted is a stool sample. Generally speaking, the higher the enrichment of Enterobacteriaceae in the test results, the worse the treatment effect and/or prognosis, and the lower the enrichment of Enterobacteriaceae, the better the treatment effect and/or prognosis. Prognosis. The evaluation of the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke specifically refers to evaluating the early recovery outcome and/or long-term functional outcome of ischemic stroke patients. The early recovery outcome can be measured by the NIHSS score. For example, the higher the NIHSS score improvement ratio, the case can be considered to have good early recovery, and vice versa, the case can be considered to have poor early recovery. In a specific embodiment of the present invention, , an improvement of NIHSS score of ≥40% can be considered as a case with good early recovery, while an improvement of NIHSS score of <40% can be considered as a case of poor early recovery. The long-term functional outcome can be measured by the Rankin Scale (mRS) score, which provides a clinical disability score that reflects lifestyle and independent living ability. For example, a lower score indicates a good long-term functional outcome. , otherwise it indicates poor long-term functional outcome. In a specific embodiment of the present invention, the range is from 0 (no symptoms) to 6 (death). A score of 0 or 1 by the evaluator indicates optimal functional outcome, and a score of 0-2 indicates good or functionally independent (good) outcome.

In the present invention, the kit can be used to evaluate the therapeutic effect of ischemic stroke treated by various conventional treatment methods for ischemic stroke and/or to judge the prognosis of ischemic stroke. Treatment methods can be anti-platelet, improve circulation, nourish nerves, regulate blood lipids, blood pressure, blood sugar and other symptomatic treatments.

In the present invention, it is preferable to use substances for detecting Enterobacteriaceae and fasting blood glucose in preparing a kit for evaluating the therapeutic effect of ischemic stroke and/or judging ischemic stroke. prognosis. The substance for detecting fasting blood glucose can be various detection products in the field that can detect fasting blood glucose of patients. The combined use of Enterobacteriaceae indicators and fasting blood glucose indicators can further improve the therapeutic effect of ischemic stroke and/or the accuracy of judging the prognosis of ischemic stroke. The trend of Enterobacteriaceae indicators is as mentioned above, and For fasting blood glucose indicators, higher fasting blood glucose often corresponds to poorer ischemic stroke treatment effect and/or judgment of ischemic stroke prognosis, and conversely, it corresponds to better ischemic stroke treatment effect. and/or determine the prognosis of ischemic stroke.

A second aspect of the present invention provides the use of a substance for detecting Enterobacteriaceae in the preparation of a kit for assessing whether a subject is susceptible to ischemic stroke with a poor prognosis. The kit is used to evaluate whether a subject is susceptible to ischemic stroke with a poor prognosis based on the enrichment degree of Enterobacteriaceae in the sample. It is preferred that the sample targeted is a stool sample. Generally speaking, the higher the enrichment of Enterobacteriaceae in the test results, the subject is considered to be susceptible to ischemic stroke with a poor prognosis, while the lower the enrichment of Enterobacteriaceae, the subject is considered to be less likely to have a prognosis. Poor ischemic stroke.

In the present invention, the use of a substance for detecting Enterobacteriaceae and fasting blood glucose in preparing a kit for evaluating whether a subject is susceptible to ischemic stroke with a poor prognosis is preferred. The combined use of Enterobacteriaceae indicators and fasting blood glucose indicators can further improve the accuracy of assessing whether subjects are susceptible to ischemic stroke with poor prognosis. The trend of Enterobacteriaceae indicators is as described above, while for fasting blood glucose indicators , higher fasting blood glucose often corresponds to the assessment subject being prone to ischemic stroke with poor prognosis, and conversely, it is considered that the subject is less prone to ischemic stroke with poor prognosis.

A third aspect of the present invention provides the use of a substance for detecting Enterobacteriaceae in preparing a kit for diagnosing ischemic stroke. The kit is used to diagnose the disease type of ischemic stroke based on the enrichment degree of Enterobacteriaceae in the sample. Preferably, the sample targeted is a stool sample. Generally speaking, the higher the enrichment of Enterobacteriaceae in the test results, the diagnosis is considered to be an ischemic stroke with a poor prognosis, while the lower the enrichment of Enterobacteriaceae is, the diagnosis is a prognosis. Better ischemic stroke.

In the present invention, the use of a substance for detecting Enterobacteriaceae and fasting blood glucose in preparing a kit for diagnosing ischemic stroke is preferred. The combined use of Enterobacteriaceae indicators and fasting blood glucose indicators can further improve the accuracy of diagnosing the type of ischemic stroke. The trend of Enterobacteriaceae indicators is as mentioned above. For fasting blood glucose indicators, higher fasting blood glucose is It is considered that the diagnosis object is an ischemic stroke with a poor prognosis, and conversely, it is considered that the diagnosis object is an ischemic stroke with a better prognosis.

As mentioned above, the kit provided by the present invention can efficiently and accurately conduct risk assessment, diagnosis and prognosis of stroke, so that the intestinal microbiota can be used as a new biomarker to assess the risk of stroke and diagnose stroke. It is of great significance to identify the types of stroke and evaluate the prognosis of stroke, including early recovery outcomes and long-term functional outcomes, and can be developed as new biomarkers in precision medicine.

The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, They are not intended to limit the scope of the invention; in the specification and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field. conventional technology. These techniques have been well described in the existing literature. For details, see Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds .), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

The study cohort consisted of patients who visited the Department of Neurology, Nanfang Hospital, Southern Medical University, from February 2014 to June 2015. This study enrolled patients with newly diagnosed ischemic stroke. Ischemic stroke is defined as a clinical syndrome associated with imaging-confirmed acute infarction consistent with findings on brain imaging-magnetic resonance imaging or magnetic resonance angiography. According to the diagnostic index of ischemic stroke, all patients were diagnosed with ischemic stroke on admission. Inclusion criteria were as follows: (1) aged between 18 and 80 years old, (2) diagnosed with acute moderate to severe ischemic stroke (severity assessed by National Institutes of Health Stroke Scale [NIHSS], NIHSS score ≥ 4) , (3) admission within 7 days of ischemic stroke onset, (4) fecal samples collected within 48 hours of admission. The exclusion criteria were as follows: (1) Use of antibiotics, prebiotics or probiotics within one month before admission; (2) Admission after 7 days of stroke onset; (3) Death within 7 days of stroke onset; (4) Liver cirrhosis, renal failure and malignant tumors and other systemic diseases. For validation purposes, an independent cohort was recruited from March 2016 to December 2017 using the same selection criteria. The Ethics Committee of Southern Medical University approved all aspects of this study, and informed consent for data collection was obtained from all subjects or their legal guardians.

Patient demographics: clinical characteristics include NIHSS score (National Institutes of Health Stroke Scale) on days 0 and 7 of admission, age (Age), gender (sex), smoking history (smoke) ), history of alcohol consumption (alcohol), history of medication (medicine), history of stroke (History of Stroke), history of hypertension (History of HBP), history of coronary heart disease (History of CAD), history of diabetes (History of DM), glycated hemoglobin (glycatedhemoglobin) ,HbAlc), blood sugar (GLU,glucose), hyperlipidemia (HLP,hyperlipidemia), total cholesterol (TC,Total cholesterol), very low-density lipoprotein (VLDL,Verylow-density lipoprotein), high-density lipoprotein (HDL) , high-density lipoprotein), triglycerides (TG, Triglycerides), low-density lipoprotein (LDL, low-density lipoprotein), creatinine (Cr, creatinine), uric acid (UA, Uric Acid), white blood cells (WBC, white blood cell). In addition to the above data, the patient’s completed cranial imaging and blood vessel examination results will also be collected. Collect fecal samples from patients within 48 hours after admission. After collection, the fecal samples are quickly placed in ice boxes and transferred to the laboratory. High-pressure sterile EP tubes are used for packaging. The packaging console is a sterilized fume hood. Label the aliquots and freeze them in a -80°C refrigerator within 2 hours of collection. After determining the enrolled samples, conduct post-operative analysis.

The person in charge of data collection is a trained graduate student, and after two people randomly select part of the data to verify that it is correct, the data will be analyzed later.

Example 1

Stroke scale score:

The National Institute of Health Stroke Scale (NIHSS) was used to evaluate neurological impairment, score according to the table, and record the results for 2 minutes. Except for necessary guidance, the subject cannot be reminded repeatedly. The evaluators have all received specialized clinical evaluation training, and the diagnosis is determined by an associate chief physician who has been engaged in clinical neurology for at least 5 years.

Microbiological studies of fecal samples:

Extraction of total DNA from stool samples (total DNA of intestinal flora):

In this study, the Mo Bio PowerSoil DNA Extraction Kit was used to extract total bacterial DNA from fecal samples. The extraction process was carried out strictly in accordance with the instructions of the Mo Bio kit. The extraction product of total bacterial DNA was stored in a -20°C refrigerator for subsequent PCR operations to amplify the target genes.

PCR amplification of bacterial 16S rRNA V4 region gene characteristic tag sequence:

After obtaining the total bacterial DNA products of all stool samples, universal primers with barcodes in the 16s rRNA V4 variable region were used for the next step of the PCR amplification process. The PCR cycle parameter settings are shown in Table 1 below:

Table 1

The PCR system was established. The system establishment process was completed under sterile conditions, and the operating platform was a biological safety cabinet. The designed capacity of the PCR reaction system is 25 μl. 25μl PCR reaction system components: 0.5μl template DNA (bacterial DNA), 1.5μl Mg2+ (25mM), 0.25μl TaKaRa Ex Taq DNA polymerase (2.5 units), 2.5μl TaKaRa 10×Ex Taq buffer (remove Mg2+) , 2.0μl dNTP mixture (2.5mM) (TaKaRa, Dalian, China), 0.5μl 10μM downstream primer (805R), 0.5μl 10μM upstream primer with barcode (514F) (GTGCCAGCMGCCGCGGTAA) (SEQ ID NO.1), downstream primer 805R (GGACTACHVGGGTWTCTAAT) (SEQ ID NO. 2) and 17.25 μl ddH2O. (All dilutions are sterile ddH2O)

After PCR amplification, agar gel electrophoresis technology is used to detect the PCR amplification product to determine whether the target band is amplified. Samples that successfully amplify the target band can be sent for sequencing.

Sequencing and bacterial community data analysis:

All PCR products that successfully amplified the target band were quantitatively mixed at a concentration of 100ug/L and sent to BGI (Shenzhen, China) (stored under frozen conditions). Illumina Miseq (PE 150) sequencing technology was used for bacterial analysis. Group gene sequencing. Use BIPES technology sequencing to preprocess all raw sequences before sequencing (see Zhou HW, Li DF, Tam NF, et al. BIPES, a cost-effective high-throughput method for assessing microbial diversity. ISME J, 2011, 5:741 -749).

The sequencing results comprehensively use bioinformatics tools such as Mothur, QIIME, and BIPES to process the sequence, quality control, remove chimeras, splice to obtain the target tag sequence, and finally sort each sample; further, through Usearch or UPARSE clustering, further processing OTU species classification (see He Y, Caporaso JG, Jiang XT, et al. Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity[J]. Microbiome, 2015, 3:20). The final results are further obtained by corresponding alpha and beta diversity parameters, and multivariate statistical analysis such as PCoA and PCA is performed. Finally, tools such as LEfSe were used to find differences in bacterial community structure between different groups.

OTU (Operational Taxonomic Units): In biological information analysis, each sequence obtained by sequencing represents a bacterium. We need to perform a classification operation (cluster) on the sequences, classify the sequences into many groups (each group is an OTU) according to their similarity to each other (specified similarity 96%, 97% or 98%), and then proceed after the OTU is divided Only through statistical analysis of biological information can we further know the number of bacterial species, genera and other information in the sequencing results of a sample. The largest OTU sequence of Enterobacteriaceae used in the analysis process is as follows:

TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGTGGT TTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGAT CTGGAGGAATACCGGTGGCAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGA AAGCGTGGGGAGCAAACAGG(S) EQ ID NO.3)

The PyNAST algorithms are used to calibrate the sequence of each OTU, and the representative sequence of each OTU needs to be determined by the frequency of the sequence. Next, remove the chimera, insert the representative sequence of OTU into FastTree software for processing, and then generate a phylogenetic tree containing all OTU metabolic sequences. On this basis, the alpha diversity (α-diversity) and Analysis of beta diversity (β-diversity analysis).

α-diversity reflects the diversity of a microbial community, and its indicators include PD whole tree indicators (representing phylogenetic diversity); Chao1 and observed species indicators (representing species richness); Shannon index indicators (taking into account both species richness and uniformity) sexual indicators).

Principal component analysis (PCA) has been widely used to compare the community structures of different intestinal flora. Principal component analysis (PCA) uses a dimensionality reduction method based on the degree of correlation between samples to convert most indicators into several comprehensive indicators for analysis. In this study, principal coordinate analysis (PCoA), which is similar to principal component analysis (PCA), was used to conduct unweighted and weighted analysis of beta-diversity between groups among each sample. result. The PCoA analysis method starts from the clustering matrix between samples, and calibrate the samples in a new coordinate system according to the clustering distance through the rotation of the three-dimensional coordinate axis.

The LEfSe (linear discriminate analysis size effect) analysis method was used to find bacterial groups with significant differences between the control group and the disease group. In this analysis, the LDA (linear discriminant analysis) critical value was set to 2 or 4. LEfSe analysis, or LDA Effect Size analysis, is an analysis method for exploring markers of high-dimensional organisms. It can identify genomic characteristics between different biological conditions, identify differences between two or more different ecological groups, and enable comparisons between groups. In addition, subgroup comparisons within groups can also be performed to find species with significant differences in abundance between groups (i.e., biomakers). This analysis method first detects species with significant differences in abundance between different groups in multiple groups of samples (using the non-parametric factor Kruskal-Wallis rank sum test); then performs difference analysis between groups (using the Wilcoxon rank of non-parametric factor group analysis) and test); finally, the linear discriminant analysis (LDA) method is used to reduce the dimensionality of the data to help evaluate the influence of species with significant differences between groups in the differences between groups (ie, LDA score). Finally, bar graphs are used to clearly display the LEfSe analysis results. Different colors in the bar graph represent different groups, and the height of the bar graph represents the degree of bacterial difference.

Definition of early improved outcomes and long-term functional outcomes:

The percentage of NIHSS improvement on day 7 after admission was chosen as the measure of early improvement (EI) (see Kharitonova T, et al. Association of Early National Institutes of Health StrokeScale Improvement with Vessel Recanalization and Functional Outcome AfterIntravenous Thrombolysis in Ischemic Stroke.Stroke; 2011;42:1638–1643; KerrDM, et al. et al. LesionLocations Influencing Baseline Severity and Early Recovery in IschaemicStroke. Eur J Neurol. 2014; 21:1226–1232). NIHSS scores were assessed from the clinical assessment template at admission, at baseline and on day 7 of treatment, using previously established NIHSS assessment methods (see Williams LS, YilmazEY, Lopez-Yunez AM. Retrospective Assessment of Initial Stroke Severity with The NIH Stroke Scale. Stroke. 2000;31:858–862). Analysis of the International Stroke Trials Archive (VISTA) showed that the 7-day NIHSS score was the most sensitive for assessing treatment effect compared with typically recorded endpoints in acute stroke trials (30- and 90-day modified Rankin scale [mRS] and 90-day NIHSS). node. Additionally, patients with acute stroke typically remain in the hospital for nearly 7-14 days; therefore, the 7-day NIHSS score is easy to use and can be used for follow-up and future clinical management. Therefore, patients were classified into the EI (≥40% improvement in NIHSS score after 7 days of standard treatment) group or the adverse EI (poor EI outcome, <40% improvement in NIHSS score at 7 days compared with baseline) group.

Patients or their caregivers were followed by telephone 90 days after stroke, and functional outcomes were obtained using modified Rankin Scale (mRS) scores. This observational scale provides a clinical disability score that reflects lifestyle and ability to live independently. The range is from 0 (no symptoms) to 6 (death). A score of 0 or 1 by the evaluator indicates optimal functional outcome, and a score of 0-2 indicates good functional outcome or a functionally independent outcome (see Rost NS, Biffi A, Cloonan L, ChorbaJ, Kelly P, Greer D, et al. Brain Natriuretic Peptide Predicts Functional Outcome in Ischemic Stroke. Stroke. 2012; 43:441–445).

data analysis:

All statistical analyzes were performed using R3.3.2. Continuous variables were expressed as median (interquartile range, IQR) or mean ± standard deviation (SD). Categorical variables are expressed as proportions. For microbiota analysis, the Adonis test implemented in QIIME 1.9.1 was used. When analyzed as an independent variable, Enterobacteriaceae were log-transformed [lg(Enterobacteriaceae × 10 ⁵ + 1)] to quantify the association between biomarkers and EI or 90-day functional outcomes. Check the normality of the data using the Shapiro-Wilk test. Compare subjects using the t test, Mann-Whitney test, chi-square test or Fisher's exact test and Wald test where appropriate. Values of P<0.05 (two-tailed) were considered significant.

Univariable and multivariable logistic regression analyzes were used to evaluate the association between Enterobacteriaceae and adverse EI in the study cohort, as well as the association between adverse EI outcomes and 90-day functional outcomes. All variables showed correlated trends in univariate analysis (P<0.20) (Adverse EI outcome model: previous antiplatelet use, history of stroke, blood glucose, UA, HCY, total cholesterol [TC], high-density lipoprotein [HDL] ], low-density lipoprotein [LDL], and Enterobacteriaceae; best outcome model: NIHSS score, Enterobacteriaceae, and adverse EI; good functional outcome model: NIHSS score, Enterobacteriaceae, and adverse EI). The predictive performance of adverse EI and 90-day functional outcomes was assessed by comparing receiver operating characteristic (ROC) curves using the multivariable model described above. Relative risk was expressed as odds ratio (OR) with 95% confidence interval (CI).

result:

An overall summary of patient characteristics in the study and validation cohorts is provided in Table 2:

Table 2

Unless otherwise stated, data are medians (interquartile range, IQR). Percentages are shown in parentheses.

There is no significant difference in traditional parameters between EI and poor EI group:

A total of 36 patients with moderate to severe acute ischemic stroke were included in the modeling cohort to compare the microbiota between the EI and adverse EI groups. After 7 days of treatment, 15 patients (41.7%) showed NIHSS improvement ≥40% and were classified as the EI group, while the remaining 21 patients (58.3%) were classified as the poor EI group. Age, NIHSS score on admission, history of HBP or DM, and blood glucose have been reported to be potential indicators related to the prognosis of acute stroke. In this study, none of these indicators showed significant differences between the EI and poor EI groups. Specifically, as shown in Table 3.

table 3

Unless otherwise stated, data are medians (interquartile range, IQR). Percentages are shown in parentheses.

There were significant differences in the intestinal flora of the two groups, among which the Enterobacteriaceae were enriched in the intestinal tract of the adverse EI group:

Principal coordinate analysis (PCoA) was used to determine whether the bacterial community structure of the two groups was statistically different. For the unweighted UniFrac distance, the two groups were significantly different (Adonis test, R2=0.034, P=0.044) (Fig. 1A), suggesting that there are differences in intestinal flora characteristics between groups. We performed similar analyzes using other distances, including weighted UniFrac (R2 = 0.034, P = 0.29); Bray-Curtis (R2 = 0.036, P = 0.197); binary Jaccard (R2 = 0.033, P = 0.006); and Pearson (R2=0.044, P=0.139). These results suggest that the gut microbiota in the early acute phase may be used to differentiate between EI and adverse EI outcomes in stroke patients.

Then, taxonomic differences between the two groups were evaluated using LEfSe, a high-dimensional biomarker discovery algorithm. In LEfSe, higher LDA values indicate more significant differences in a certain type of bacteria between test groups. Experimentally, it can be observed that Enterobacteriaceae and Enterobacteriaceae (containing only Enterobacteriaceae) are significantly enriched in the poor EI group, while other taxa including Actinomycetes, Alphaproteobacteria, and Actinobacteria are significantly enriched in EI. group (Fig. 1B-C). Among them, Enterobacteriaceae showed the most significant difference, with an LDA value higher than 4 (Fig. 1C). We further used the Mann-Whitney test to examine the significance of the relative abundance of Enterobacteriaceae between these two groups. The relative abundance of Enterobacteriaceae was significantly increased in the poor EI group (median [interquartile range, IQR] 12.2% (2.8-29.0) vs. 4.7% (1.1-7.9); Mann-Whitney test, P = 0.027), almost 3 times that of the EI group (Fig. 1D). For other taxa identified with LEfSe, the Mann-Whitney test results were significantly different. However, the absolute abundance of other taxa was much lower than Enterobacteriaceae.

Enterobacteriaceae predict poor EI:

Univariate and multivariate analyzes were used to test whether Enterobacteriaceae can be used to predict adverse EI outcomes. The variables entered in the multivariate logistic regression model were as follows: sex, age, smoking history, blood glucose, baseline NIHSS score, stroke history, HBP, DM, and HLP, which are potential confounders that have been previously reported to be associated with acute stroke. In addition, some laboratory test values were also added to the model, and univariate and multivariate logistic regression analysis to predict adverse EI outcomes in stroke patients in the study group are shown in Table 4. In univariate analysis, Enterobacteriaceae (OR: 6.2, 95% CI: 1.27-30.34); TC (OR: 0.39, 95% CI: 0.19-0.79); HDL (OR: 0.02, 95% CI: 0- 0.44); and LDL (OR: 0.23, 95% CI: 0.07-0.68) were associated with adverse EI outcomes. Fasting plasma glucose, history of antiplatelet medication, history of stroke, UA, and HCY showed a trend toward association with adverse EI (P<0.2). In multivariable analysis adjusting for the above variables, only Enterobacteriaceae (adjusted OR: 6.27, 95% CI: 1.16-33.73) remained independently predictive of adverse EI outcome. The prediction model included Enterobacteriaceae and fasting plasma glucose, whereas UA, HCY, TC, HDL, LDL, history of stroke and history of antiplatelet drugs were removed because of multicollinear associations between these variables. The ROC plot of the logistic regression analysis showed that the predictive performance of the model (Enterobacteriaceae + fasting plasma glucose) for adverse EI outcomes was 78.8% (area under the curve [AUC] estimated at 78.8%), while the predictive performance of Enterobacteriaceae for adverse EI Performance was with similar efficiency (AUC estimate, 76.9%) (Figure 3A).

Table 4

AUC represents the area under the curve; history of antiplatelet drugs, use of antiplatelet drugs before admission; HBP, hypertension; DM, diabetes mellitus; HLP, hyperlipidemia; TC, total cholesterol; TG, triglycerides; HDL, high-density lipoprotein Protein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein; WBC, white blood cell count; Cr, creatinine; UA, uric acid; HbA1c, glycosylated hemoglobin; HCY, homocysteine; SBP, systolic blood pressure; DBP, Diastolic blood pressure; PBP, pulse pressure. *P<0.05. #Adjust all the above values in the table.

Validation of Enterobacteriaceae in predicting adverse EI:

In addition, a new clinical cohort was re-established from 2016 to 2017, including a validation cohort of 88 patients (EI group: n=37 [42.0%], poor EI group: n=51 [58.0%]).

The EI and adverse EI groups were similar in age, gender, HBP, DM, and NIHSS scores at admission (see Table 5 for analysis of EI-related adverse factors among validation cohort subgroups). Similar to the study group, the results of LEfSe analysis showed that enriched Enterobacteriaceae were always the most important differential bacteria in the poor EI group compared with the EI group (Figure 2B-C). In the Mann-Whitney test, a significant increase in Enterobacteriaceae was observed in the poor EI group compared with the EI group (median [IQR] 7.3%, 95% CI [4.0-16.4] vs. 4.1%, 95% CI[1.9-8.4]; P=0.0011) (Figure 2D). The ROC results showed that the area under the curve for Enterobacteriaceae for adverse EI outcome was 70.2%, while the predictive model for Enterobacteriaceae plus fasting glucose was 73.3% (Fig. 3B).

table 5

Unless otherwise stated, data are medians (interquartile range, IQR). Percentages are shown in parentheses.

Using adverse EI and Enterobacteriaceae to predict 90-day functional outcome:

Univariate analysis of long-term functional outcomes at 90 days after ischemic stroke showed that baseline NIHSS score, Enterobacteriaceae and poor EI were associated with best 90-day outcomes (mRS 0-1) and good outcomes in the modeling cohort (mRS 0-2) were significantly correlated (all P<0.05). The univariate and multivariate logistic regression analyzes used to predict the 90-day functional outcomes of the patients in the study cohort are shown in Table 5. The prediction of 90-day functional outcomes after ischemic stroke by adjusted adverse EI outcomes for the validation cohort and the entire cohort is shown in Table 7. In multivariable analysis adjusted for the above variables and relevant confounders, adverse EI (OR: 0.059 , 95% CI: 0.005-0.656) and NIHSS score (OR: 0.287, 95% CI: 0.097-0.846) can independently predict the best 90-day outcome (mRS 0-1). We removed Enterobacteriaceae from the model because it is linearly associated with poor EI. Similarly, adverse EI outcome (OR: 0.005, 95% CI: 0-0.392) and NIHSS score (OR: 0.372, 95% CI: 0.179-0.770) were independent predictors of favorable 90-day outcome (mRS 0-2).

Adding adverse EI outcomes to models predicting optimal and favorable outcomes at 90 days significantly increased their predictive performance (AUC estimates increased from 0.821 to 0.919 [P=0.024] and from 0.786 to 0.925 [P=0.018], respectively) ) (Figure 4A).

In the validation cohort, adverse EI remained an independent predictor of optimal 90-day outcome and favorable outcome. The addition of adverse EI outcomes increased the predictive performance of best and good 90-day outcomes (AUC estimates increased from 0.747 to 0.802 [P=0.006] and from 0.802 to 0.839 [P=0.016], respectively) in the validation cohort. confirmed (Figure 4B).

In the entire large cohort (i.e., modeling + validation cohort), Enterobacteriaceae was associated with the best 90-day outcome (OR: 0.249, 95% CI: 0.089-0.693) and good outcome (OR: 0.320, 95% CI: 0.122) -0.844) independent predictor. The addition of Enterobacteriaceae also increased the predictive performance of optimal and favorable outcomes at 90 days in the entire patient cohort (AUC estimates increased from 0.764 to 0.802 [P=0.008] and from 0.800 to 0.825 [P=0.021], respectively) (Figure 4C).

Table 6

*P<0.05. #Adjust all the above values in the table.

Table 7

Multivariate logistic regression included age, gender, baseline NIHSS, history of HBP, history of DM, history of HLP, history of CAD, and smoking history. ^* P<0.05; ^** P<0.01; ^*** P<0.001.

The entire cohort, which is the sum of the modeling and validation cohorts.

It can be seen from the above experimental results that intestinal Enterobacteriaceae in the early acute phase after ischemic stroke are closely related to 7-day adverse EI outcomes, and traditional clinical variables have low effectiveness in predicting 7-day adverse EI outcomes. Furthermore, intestinal Enterobacteriaceae in the early acute phase can be an independent predictor of 90-day functional outcome after ischemic stroke. The study results confirmed that adverse EI results at 7 days are independent predictors of 90-day functional outcomes and can improve the predictive performance of 90-day functional outcomes after ischemic stroke.

As mentioned above, EI is positively correlated with long-term recovery outcomes, and improvement of neurological function is crucial for clinical management, but not all patients can achieve satisfactory recovery through previous standardized treatments. Identifying patients at higher risk for adverse EI is important for physicians to adjust treatment plans or develop new treatments. In fact, EI is not a very precise term clinically. Although the NIHSS score is widely used, the "early" time period usually ranges from 2 hours to 30 days from onset. In this study, baseline and day 7 NIHSS scores were used for assessment. The median length of stay (LOHS) for acute stroke patients is usually 7 to 14 days. The seven-day follow-up is a time point that reflects the trend of early recovery. It is the largest proportion of recovery status per time period and is a strong indicator for predicting final outcome. . Patients usually reach a relatively stable state on the 7th day after onset, with almost half of them discharged from the hospital around this time. Furthermore, studies have shown that this metric has better value in acute stroke trials than other typical endpoints (mRS score at 30 and 90 days, NIHSS score at 90 days). To assess early improvement or final functional outcome, demographic and clinical measures including baseline NIHSS score, age, stroke severity, lesion location, previous ischemic stroke, uric acid, and diabetes have demonstrated variable accuracy. A model integrating age at admission and NIHSS score correctly identified 62.9% of patients with incomplete recovery or death and 83.2% of patients with complete recovery 100 days after acute stroke. However, results for these measures have been reported to be inconsistent, and their power to predict early outcomes in this study was poor.

The inventor of the present invention found that intestinal flora, especially Enterobacteriaceae, is an independent predictor of the prognosis of acute ischemic stroke. By using 4 different statistical methods - LEfSe analysis (where LDA value is 4), Mann-Whitney U test, univariate and multivariate logistic regression analysis - we confirmed significant differences in Enterobacteriaceae between EI and poor EI groups and the impact of Enterobacteriaceae on adverse EI outcomes with reliable predictive performance in two independent cohorts (AUC 76.9% and 70.2%, respectively). Negative results with traditional measures may be attributed to the heterogeneity of the ischemic stroke population, as most patients receive appropriate medications at admission. In contrast, to date, there are no clinical treatments targeting the gut microbiota during hospitalization for acute stroke patients. The study data further showed that the disordered intestinal flora remained relatively stable during the patient's hospitalization. Changes in the gut microbiota, including an increase in Enterobacteriaceae, are observed days after stroke (1-4) and persist without significant changes for at least 3 weeks. Therefore, the gut microbiota in the acute phase can be a reliable biomarker for outcome prediction.

To sum up, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

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<110> Zhujiang Hospital of Southern Medical University

Nanfang Hospital of Southern Medical University

<120> Use of Enterobacteriaceae as biomarkers for ischemic stroke

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Claims (7)

1. Use of a substance for detecting enterobacteriaceae in the preparation of a kit for assessing the therapeutic effect of ischemic stroke and/or for judging the prognosis of ischemic stroke;

wherein the substance for detecting enterobacteriaceae is a substance for detecting the enrichment degree of enterobacteriaceae;

the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in a fecal sample; and/or the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in the intestinal tract;

ischemic stroke is a moderately severe stroke;

assessing the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke refers to assessing the early recovery outcome and/or the long-term functional outcome of an ischemic stroke patient, said early recovery outcome being measured by NIHSS score and said long-term functional outcome being measured by rank scale (mRS) score;

NIHSS score improvement is greater than or equal to 40% of cases with good early recovery, while NIHSS score improvement is less than 40% of cases with poor early recovery, the rank scale (mRS) score ranges from 0 to 6, if the evaluator judges 0 or 1, the functional result is the best, if the score is 0-2, the functional result is good or independent.

2. The use according to claim 1, wherein the substance for detecting enterobacteriaceae is a substance for detecting bacterial 16S rRNA.

3. The use according to claim 2, wherein the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in the intestinal tract of the large intestine;

and/or, the substance for detecting enterobacteriaceae is a substance for detecting bacterial 16S rRNA V4 region.

4. The use according to claim 1, wherein the sample is a fecal sample.

5. The use according to claim 1, wherein the ischemic stroke is NIHSS not less than 4.

6. The use of claim 1, wherein the ischemic stroke is selected from the group consisting of total stroke;

and/or, the ischemic stroke is an acute phase.

7. Use of a substance for detecting enterobacteriaceae in the preparation of a kit for assessing whether a subject is susceptible to ischemic stroke with poor prognosis;

wherein the substance for detecting enterobacteriaceae is a substance for detecting the enrichment degree of enterobacteriaceae;

the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in a fecal sample; and/or the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in the intestinal tract;

ischemic stroke is a moderately severe stroke;

the higher the enrichment degree of enterobacteriaceae in the result of detecting the enrichment degree of enterobacteriaceae, the corresponding subject is ischemic cerebral apoplexy with poor prognosis, and the lower the enrichment degree of enterobacteriaceae, the subject is considered to be ischemic cerebral apoplexy with better prognosis.