CN110964778B - Use of enterobacteriaceae as ischemic cerebral apoplexy biomarker - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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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
Technical Field
The invention relates to the field of biomarkers, in particular to application of enterobacteriaceae as an ischemic cerebral apoplexy biomarker.
Background
The risk of identifying poor stroke outcomes is important to clinical management, but not all patients can get satisfactory recovery through past standardized treatments. For doctors and patients, it is important for doctors to adjust treatment plans or develop new treatment methods to identify patients at higher risk of undesirable EI.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide the use of Enterobacteriaceae (Enterobacteriaceae) as ischemic stroke biomarker for solving the problems in the prior art.
To achieve the above and other related objects, the present invention provides in one aspect the use of a substance for detecting enterobacteriaceae in the preparation of a kit for evaluating the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke.
In some embodiments of the invention, the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in a fecal sample.
In some embodiments of the invention, the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in the intestinal tract.
In some embodiments of the invention, the substance for detecting enterobacteriaceae is a substance for detecting the degree of enrichment of enterobacteriaceae.
In some embodiments of the invention, the agent for detecting enterobacteriaceae is an agent that detects bacterial 16S rRNA.
In some embodiments of the invention, the substance for detecting enterobacteriaceae is a substance for detecting enterobacteriaceae in the intestinal tract of the large intestine.
In some embodiments of the invention, the agent for detecting enterobacteriaceae is an agent that detects the 16s rRNA V4 region of bacteria.
In some embodiments of the invention, the kit evaluates the therapeutic effect of ischemic stroke and/or determines the prognosis of ischemic stroke based on the degree of enrichment of enterobacteriaceae in the sample.
In some embodiments of the invention, the sample is a fecal sample.
In some embodiments of the invention, the ischemic stroke is NIHSS.gtoreq.4.
In some embodiments of the invention, the ischemic stroke is selected from the group consisting of total stroke.
In some embodiments of the invention, the ischemic stroke is acute phase.
In some embodiments of the invention, the assessing the therapeutic effect of ischemic stroke and/or the prognosis of ischemic stroke specifically refers to assessing the early recovery outcome and/or the long-term functional outcome of an ischemic stroke patient.
In another aspect, the 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.
In another aspect, the invention provides the use of a substance for detecting enterobacteriaceae in the preparation of a kit for diagnosing ischemic stroke.
Drawings
FIG. 1 shows a comparison of the population of EI with a subset of undesirable EI in a modeled queue according to the present invention. (A) The PCoA plot shows a significant difference in bacterial colony between the EI group and the poor EI group. (B) Mean relative abundance of major bacterial groups of the families, phylum and genus of EI and ill EI patients. (C) LEfSe determines the taxonomic group with the greatest difference between the two groups. Only the enterobacteriaceae and enterobacteriaceae groups have an LDA effective threshold >4. (D) Box plots show the abundance of enterobacteriaceae in poor EI groups compared to EI groups. PC, principal coordinate analysis (PCoA). EI, EI patient (n=15); patients with poor EI and no EI (n=21).
FIG. 2 is a schematic representation of the comparison of a microbial community of EI with a sub-population of undesirable EI in a validation queue according to the invention, wherein (A) the PCoA graph shows that there is no significant difference in bacterial community between the EI group and the undesirable EI group in the validation queue; (B) Average relative abundance of major bacterial groups at the scientific level for EI and poor EI patients; (C) LEfSe identifies the taxonomic group between the two groups that differs the most. Only gamma-Proteobacteria, enterobacteriaceae, proteobacteria classification group meets LDA effective threshold >4; (D) Box plots show the abundance of enterobacteriaceae in poor EI groups compared to EI groups. PC, principal coordinate analysis (PCoA). EI, EI patient (n=37); patients with poor EI and no EI (n=51).
FIG. 3 shows a graph of predicted performance of the poor EI outcome of the invention, wherein (A) the predicted performance of the model (Enterobacteriaceae+fasting glucose) on the poor EI outcome was 78.8% (area under the curve [ AUC ] estimated to be 78.8%) in the modeled cohort, while the predicted performance of the Enterobacteriaceae on the poor EI had similar efficiency (AUC estimated, 76.9%); (B) In the validation cohort, the area under the curve of enterobacteriaceae at poor EI end was 70.2%, while the predictive model of enterobacteriaceae plus fasting blood glucose was 73.3%.
FIG. 4 shows a schematic representation of a model of the present invention for predicting 90-day best and good outcomes, wherein (A) adding a poor EI outcome to the model for predicting 90-day best and good outcomes significantly increases their predictive performance; (B) Addition of poor EI outcomes increases the 90 day best outcome and addition of good outcomes (C) enterobacteriaceae also increases the predictive performance of the 90 day best outcome and good outcomes in the overall patient cohort (pooled modeling and validation cohort).
Detailed Description
The inventor of the present invention has found that, through a great deal of research, enterobacteriaceae has a close relationship with the diagnosis and treatment effects of ischemic stroke and/or the prognosis of ischemic stroke, and has completed the present invention on the basis of this.
In a first aspect the invention provides the use of a substance for detecting enterobacteriaceae in the preparation of a kit for assessing the therapeutic effect of and/or for judging the prognosis of an ischemic stroke. The ischemic cerebral apoplexy refers to the general term of brain tissue necrosis caused by cerebral blood supply arterial occlusion and insufficient blood supply, and the vascular occlusion can be cerebral embolism, cerebral thrombosis and the like.
Cerebral apoplexy mainly comprises hemorrhagic cerebral apoplexy and ischemic cerebral apoplexy. Wherein ischemic cerebral apoplexy accounts for 60-70% of the strokes. Ischemic stroke mainly includes: transient Ischemic Attack (TIA); complete Stroke (CS). In one embodiment of the invention, it is preferable to target full stroke. In another embodiment of the present invention, the ischemic stroke is a moderately severe stroke patient (NIHSS score. Gtoreq.4, NIHSS (national institute of health Stroke Scale, national Institute of Health stroke scale) scoring method can be found in Williams LS, YIlmaz EY, lopez-Yonez AM. Retroselect Assessment of Initial Stroke Severity with The NIH Stroke Scale. Stroke.2000; 31:858-862). In another embodiment of the invention, the ischemic stroke is in an acute phase, which is typically within 7 days of onset.
In the present invention, the substance for detecting enterobacteriaceae may be various detection products suitable for detecting enterobacteriaceae in the field, for example, may be a PCR kit, a FISH kit, etc., and the object to which the detection product is directed may be a fecal sample, so as to better reflect the intestinal flora of the large intestine, or may be 16S rRNA of bacteria, preferably may be 16S rRNA V4 region of bacteria, so as to obtain information of 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 a fecal sample. In another embodiment of the present invention, the substance for detecting enterobacteriaceae may be a substance for detecting enterobacteriaceae in the intestinal tract, preferably the large intestinal tract, and the sample to be detected is usually a sample reflecting enterobacteriaceae in the intestinal tract of a patient. In another embodiment of the present invention, the substance for detecting enterobacteriaceae may be a substance for detecting the degree of enrichment of enterobacteriaceae, which may be the content of enterobacteriaceae in a certain amount of the sample (for example, unit volume, unit mass, etc.), the percentage of the enterobacteriaceae in the flora, etc. In another embodiment of the present invention, the method for using the substance and/or kit for detecting enterobacteriaceae may comprise the steps of: and (3) obtaining total DNA of intestinal flora in the sample, amplifying a bacterial 16S rRNA V4 region gene characteristic tag sequence, obtaining enrichment information of enterobacteriaceae in the sample according to an amplification result, and dividing bacterial types by adopting an OTU (Operational Taxonomic Units, classification operation unit) method.
In the invention, the kit is used for evaluating the treatment effect of the ischemic cerebral apoplexy and/or judging the prognosis of the ischemic cerebral apoplexy according to the enrichment degree of enterobacteriaceae in the sample, and the sample is preferably a fecal sample. Generally, the higher the degree of enrichment of the enterobacteriaceae in the test results, the poorer the therapeutic effect and/or prognosis, and the lower the degree of enrichment of the enterobacteriaceae, the better the therapeutic effect and/or prognosis. The evaluation of the therapeutic effect of ischemic stroke and/or the judgment of the prognosis of ischemic stroke specifically refers to the evaluation of the early recovery outcome and/or the long-term functional outcome of an ischemic stroke patient. The early recovery outcome may be measured by the NIHSS score, e.g., the higher the rate of improvement in the NIHSS score, the more cases that are considered to be early recovery good, and vice versa, in one embodiment of the invention, an improvement in the NIHSS score of > 40% may be considered to be early recovery good, while an improvement in the NIHSS score of <40% may be considered to be early recovery poor. The long-term functional outcome may be measured by a rank scale (mRS) score that provides a clinical disability score that reflects lifestyle and independent lifestyle, e.g., the lower the score, the better the long-term functional outcome and conversely the poor the long-term functional outcome, ranging from 0 (asymptomatic) to 6 (dead) in one embodiment of the invention. If the evaluator determines 0 or 1, this indicates that the functional result is optimal, and if the score is 0-2, this indicates a good or functionally independent (good) outcome.
In the invention, the kit can be used for evaluating the treatment effect of ischemic cerebral apoplexy treated by various conventional treatment methods aiming at the ischemic cerebral apoplexy and/or judging the prognosis of the ischemic cerebral apoplexy, wherein the treatment methods can be antiplatelet, circulatory improvement, vegetative nerve, blood fat regulation, blood pressure, blood sugar and other symptomatic treatment methods.
In the present invention, it may be preferable that the substance for detecting enterobacteriaceae and fasting blood glucose is used for preparing a kit for evaluating the therapeutic effect of ischemic stroke and/or judging the prognosis of ischemic stroke. The substance for detecting fasting blood glucose may be various detection products in the art that can detect fasting blood glucose of a patient. The combined use of the enterobacteriaceae index and the fasting blood glucose index can further improve the treatment effect on ischemic cerebral apoplexy and/or the accuracy of prognosis of ischemic cerebral apoplexy, the trend of the enterobacteriaceae index is as described above, and for the fasting blood glucose index, higher fasting blood glucose usually corresponds to poorer treatment effect on ischemic cerebral apoplexy and/or prognosis of ischemic cerebral apoplexy, otherwise, corresponds to better treatment effect on ischemic cerebral apoplexy and/or prognosis of ischemic cerebral apoplexy.
In a second aspect, the 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 for evaluating whether a subject is susceptible to ischemic cerebral apoplexy with poor prognosis according to the enrichment degree of enterobacteriaceae in the sample, and the sample is preferably a fecal sample. In general, the higher the degree of enrichment of enterobacteriaceae in the detection results, the more likely the subject is to develop ischemic stroke with poor prognosis, while the lower the degree of enrichment of enterobacteriaceae, the less likely the subject is to develop ischemic stroke with poor prognosis.
In the present invention, it may be preferable that the substance for detecting enterobacteriaceae and fasting blood glucose is used for preparing a kit for evaluating whether a subject is susceptible to ischemic stroke with poor prognosis. The combined use of the enterobacteriaceae index and the fasting blood glucose index can further improve the accuracy of assessing whether the subject is susceptible to ischemic stroke with poor prognosis, the trend of the enterobacteriaceae index is as described above, and for the fasting blood glucose index, higher fasting blood glucose usually corresponds to assessing the subject to ischemic stroke with poor prognosis, otherwise, the subject is considered to be not susceptible to ischemic stroke with poor prognosis.
In a third aspect the invention provides the use of a substance for detecting enterobacteriaceae in the preparation of a kit for diagnosing ischemic stroke. The kit is used for diagnosing the disease type of ischemic cerebral apoplexy according to the enrichment degree of enterobacteriaceae in the sample, and the sample is preferably a fecal sample. In general, the higher the degree of enrichment of enterobacteriaceae in the detection result, the less prognostic ischemic stroke is considered as the diagnosis subject, while the lower the degree of enrichment of enterobacteriaceae, the more prognostic ischemic stroke is considered as the diagnosis subject.
In the present invention, it may be preferable that the substance for detecting enterobacteriaceae and fasting blood glucose is used for preparing a kit for diagnosing ischemic stroke. The combined use of the enterobacteriaceae index and the fasting blood glucose index can further improve the accuracy of diagnosing the variety of ischemic cerebral apoplexy, the trend of the enterobacteriaceae index is as described above, and for the fasting blood glucose index, the higher fasting blood glucose is considered to be the ischemic cerebral apoplexy with poor prognosis, otherwise, the diagnosis object is considered to be the ischemic cerebral apoplexy with better prognosis.
As described above, the kit provided by the invention can efficiently and accurately perform risk assessment, diagnosis and prognosis on the stroke, so that the intestinal microbiota can be used as a novel biomarker for assessing the risk of the stroke, diagnosing the type of the stroke, assessing the prognosis of the stroke and the like, and the kit is very significant in terms of early recovery ending and long-term function ending, and can be developed into novel biomarkers in accurate medicine.
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. 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 to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art literature and see, in particular, sambrook et al MOLECULAR CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989and Third edition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman 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.
Study cohorts were patients with neurological visits in southern hospitals at southern medical universities during 2 months 2014 to 6 months 2015. The study recruited patients newly diagnosed with ischemic stroke. Ischemic stroke is defined as a clinical syndrome associated with imaging-confirmed acute infarction, consistent with the results of brain imaging-magnetic resonance imaging or magnetic resonance angiography. All patients at admission were diagnosed with ischemic stroke based on the ischemic stroke diagnostic index. Inclusion criteria were as follows: (1) between 18 and 80 years of age, (2) diagnosis of acute moderately severe ischemic stroke (national institute of health stroke scale [ NIHSS ] to assess the severity of the disease, NIHSS score > 4), (3) admission within 7 days after onset of ischemic stroke, (4) collection of stool samples within 48 hours after admission. The exclusion criteria were as follows: (1) Antibiotics, prebiotics or probiotics are used within one month prior to admission; (2) admission is made 7 days after the onset of cerebral apoplexy; (3) death within 7 days after stroke onset; (4) History of systemic diseases such as liver cirrhosis, renal failure and malignant tumor. For verification purposes, a separate cohort was recruited from month 2016, 3, to month 2017, 12 using the same selection criteria. The ethics committee of southern medical university approved all aspects of the study and informed consent for data collection was obtained from all subjects or legal guardians thereof.
Demographics of patients: clinical features include NIHSS score (National Institutes of Health Stroke Scale, national institutes of health score scale), age (Age), sex (sex), smoking History (size), drinking History (alcohol), medical History (media), stroke History (History of Stroke), hypertension History (History of HBP), coronary disease History (CAD), diabetes History (History of DM), glycosylated hemoglobin (HbAlc), glucose (GLU), hyperlipidemia (HLP), total cholesterol (TC, total cholesterol), very low density lipoprotein (VLDL, very low-density lipoprotein), high density lipoprotein (HDL, high-density lipoprotein), triglycerides (TG, triglylcerides), low density lipoprotein (LDL, low-density lipoprotein), creatinine (Cr, high Acid), uric Acid (UA, uic Acid), white blood cells (wbcell). In addition to the above data, the results of the completed examination of the patient's skull, blood vessels, etc. are also collected. The fecal sample is collected within 48 hours after the patient is admitted, and after the fecal sample is collected, the fecal sample is quickly placed in an ice box and transferred to a laboratory, and is split-packed by adopting a high-pressure sterile EP pipe, a split-packing operation table is a sterilized fume hood, marks are made after split-packing, and the fecal sample is frozen in a refrigerator at-80 ℃ within 2 hours after collection. After the group-entering samples are determined, a post-operation analysis is performed.
The data acquisition is responsible for a trained researcher, and after 2 people randomly extract part of data for verification, the data enter into later data analysis.
Example 1
Stroke scale score:
impaired neurological function was evaluated using the national institutes of health stroke scale (National Institute of Health stroke scale, NIHSS), scored on a table, and the results recorded for 2 minutes. The person to be tested cannot be repeatedly reminded except for the necessary pointing. The evaluation staff are trained in clinical specialization evaluation, and diagnosis is determined by a minor physician who engages in neurology clinic for at least 5 years.
Microbiology study of stool samples:
fecal sample total DNA (intestinal flora total DNA) extraction:
in this study, mo Bio strong soil DNA extraction kit (the Mo Bio PowerSoil DNA Extraction Kit) was used to extract bacterial total DNA in fecal samples. The extraction procedure was strictly according to the instructions of the Mo Bio kit. The extracted product of the total DNA of the bacteria is placed in a refrigerator at the temperature of minus 20 ℃ for freezing and preserving for amplifying target genes by performing PCR operation in the later period.
PCR amplification of bacterial 16S rRNA V4 region gene characteristic tag sequence:
after obtaining the bacterial total DNA product of all fecal samples, the next PCR amplification procedure was performed using universal primers with 16s rRNA V4 variable region with barcode. The PCR cycle parameters were set as shown in table 1 below:
TABLE 1
And (3) establishing a PCR system, wherein the system establishment process is completed under the aseptic condition, and the operation table is a biological safety cabinet. The design capacity of the PCR reaction system was 25. Mu.l. 25 μl of PCR reaction system components: 0.5. Mu.l template DNA (bacterial DNA), 1.5. Mu.l Mg2+ (25 mM), 0.25. Mu. l TaKaRa Ex Taq DNA polymerase (2.5 units), 2.5. Mu.l TaKaRa 10X Ex Taq slow release (de Mg2+), 2.0. Mu.l dNTP mix (2.5 mM) (TaKaRa, dalian, china), 0.5. Mu.l 10. Mu.M downstream primer (805R), 0.5. Mu.l 10. Mu.M upstream primer with barcode (514F) (GTGCCAGCMGCCGCGGTAA) (SEQ ID NO. 1), downstream primer 805R (GGACTACHVGGGTWTCTAAT) (SEQ ID NO. 2) and 17.25. Mu.l ddH2O. (all dilutions were sterile ddH 2O)
After PCR amplification, the detection of PCR amplified products is carried out by the agarose gel electrophoresis technology, and whether the target band is amplified is determined. Samples of the target band that were successfully amplified may be sequenced.
Sequencing and flora data analysis:
all PCR products of the target band are amplified successfully, mixed quantitatively according to the concentration of 100ug/L, and uniformly sent to Hua Dada gene company (Shenzhen, china) (stored in freezing condition), and the sequencing technology of Illumina Miseq (PE 150) is adopted for sequencing the flora genes. All original sequences before sequencing were pre-processed using BIPES technology 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).
Comprehensively adopting bioinformatics tools such as Mothur, QIIME, BIPES and the like to process sequences, control quality, remove chimeras, splice to obtain target tag sequences, and finally sorting each sample; further, OTU species classification was further performed by Usearch or UPARSE clustering (see He Y, caporaso JG, jiang XT, et al stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity [ J ]. Microbiolome, 2015, 3:20). And the final result further obtains corresponding alpha and beta diversity parameters, and performs PCoA, PCA and other multivariate statistical analysis. And finally, searching for the flora structure difference among different groups by combining and applying tools such as LEfSe and the like.
OTU (Operational Taxonomic Units, sorting operation unit): in biological information analysis, each sequence obtained by sequencing represents a single bacterium. The sequences are classified into a plurality of groups (each group is an OTU) according to the similarity (designated similarity 96%, 97% or 98%), the OTU is classified, and then the biological information statistical analysis is performed to obtain the information of the number of strains, bacteria and the like in a sequencing result, and the maximum OTU sequence of enterobacteriaceae used in the analysis process is as follows:
TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGTGGT TTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGAT CTGGAGGAATACCGGTGGCAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGA AAGCGTGGGGAGCAAACAGG(SEQ ID NO.3)
the sequences of each OTU are aligned using the PyNAST algorithm (PyNAST algorithms), and the representative sequence of each OTU needs to be determined by the frequency of the sequence. Next, the chimera is removed, the representative sequence of OTU is inserted into FastTree software for processing, and then phylogenetic tree containing all OTU metabolic sequences is generated, and analysis of alpha diversity (alpha-diversity) and beta diversity (beta-diversity analysis) of the flora is performed on the basis.
The alpha-diversity reflects the diversity of a microbial community, and the indexes of the alpha-diversity comprise PD whole tree indexes (representing phylogenetic diversity); chao1 and observed specie index (representing species richness); shannon index (an index that considers both species abundance and uniformity).
Principal component analysis (Principal component analysis, PCA), a method which has been widely used for comparison of different intestinal flora structures. The principal component analysis PCA is to analyze a plurality of indexes into a plurality of comprehensive indexes by adopting a dimension reduction method according to the correlation degree among samples. In this study, a principal coordinate analysis method (principal coordinate analysis, PCoA) similar to principal component analysis PCA was used to analyze the results of inter-group beta-diversity by non-weighted (unweighted) and weighted (weighted) analysis of each sample. The PCoA analysis method starts from a clustering matrix among samples, and samples are calibrated in a new coordinate system according to clustering distances through rotation of three-dimensional coordinate axes.
The LEfSe (linear discriminate analysis size effect) assay was used to find out bacterial populations with significant differences between the control and disease groups. In this analysis, an LDA (linear discriminant analysis) critical value is set to 2 or 4. The LEfSe analysis, i.e., the LDA Effect Size analysis, is an analytical method that explores for labeling of high vitamins. The method can identify the characteristics of genomics among different biological conditions, distinguish the differences among two or more different ecological groups and realize the comparison among groups. In addition, internal subgroup comparisons of the subgroups can be made to find species (i.e., boom) that differ significantly in abundance between the subgroups. The analysis method comprises the steps of firstly detecting species with obvious abundance differences among different groups in a plurality of groups of samples (adopting a nonparametric factor Kruskal-Wallis rank sum test); then performing inter-group variance analysis (Wilcoxon rank sum test using non-parametric factor group analysis); finally, the data were dimensionalized using a Linear Discriminant Analysis (LDA) method to help assess the impact of species with significant inter-group differences in the inter-group differences (i.e., LDA score). Finally, the results of the LEfSe analysis are clearly shown using bar charts. The different colors in the bar represent different groupings and the height of the bar indicates the magnitude of the degree of bacterial differentiation.
Definition of early improvement outcome and long term functional outcome:
the percentage of NIHSS improvement on day 7 post admission was chosen as a measure of Early Improvement (EI) (see Kharitonova T, et al Association of Early National Institutes of Health Stroke Scale Improvement with Vessel Recanalization and Functional Outcome After Intravenous Thrombolysis in Ischemic Stroke. Stroke; 2011;42:1638-1643;Kerr DM,et al.Seven-Day NIHSS Is A Sensitive Outcome Measure for Exploratory Clinical Trials in Acute Stroke: evidence from the Virtual International Stroke Trials Archive. Stroke.2012;43:1401-1403;Bentley P,et al.Lesion Locations Influencing Baseline Severity and Early Recovery in Ischaemic Stroke.Eur J Neurol.2014;21:1226-1232). NIHSS scores were assessed from clinical assessment templates at admission, baseline levels and day 7 of treatment using previously established NIHSS assessment methods (see Williams LS, YIlmaz EY, lopez-Yenz AM. Retroactive Assessment of Initial Stroke Severity with The NIH Stroke Scale. Stroke.2000; 31:858-862). International stroke test profile (VISTA) analysis showed that the 7-day NIHSS score was the most sensitive node to evaluate treatment efficacy compared to the typical recorded endpoints in acute stroke tests (30 and 90-day modified rank scale [ mRS ] and 90-day NIHSS). Furthermore, acute stroke patients are typically hospitalized in hospitals for approximately 7-14 days; thus, the 7 day NIHSS score is convenient to use and can be used for follow-up and future clinical management references. Thus, patients were classified as either an EI (NIHSS score improved by ≡40% after 7 days of standard treatment) group or a poor EI (poor EI outcome, <40% improvement from baseline) group by 7 days of NIHSS.
Patients or their caregivers were followed by telephone 90 days after stroke, and functional outcomes were obtained by using a modified rank scale (mRS) score. The observation scale provides clinical disability scores reflecting lifestyle and independent lifestyle capabilities. Ranging from 0 (asymptomatic) to 6 (dead). If the evaluator judges 0 or 1, this indicates that the functional result is optimal, and if the score is 0-2, this indicates that the functional result is good or a functionally independent outcome (see, rost NS, biffi A, cloonan L, chorba J, kelly P, greer D, et al, brain Natriuretic Peptide Predicts Functional Outcome in Ischemic Stroke.2012; 43:441-445).
Data analysis:
all statistical analyses were performed using R3.3.2. The continuous variable is expressed as median (interquartile range, IQR) or mean ± Standard Deviation (SD). The classification variables are expressed in scale. For microbiota analysis, the Adonis test performed in QIIME 1.9.1 was used. When analyzed as independent variables, the enterobacteriaceae were subjected to logarithmic transformation [ lg (enterobacteriaceae. Times.10) 5 + 1)]To quantify the association between the biomarker and EI or 90 day functional outcome. The data were checked for normality using the Shapiro-Wilk test. Subjects were compared using the t-test, mann-Whitney test, chi-square test or Fisher precision test, and the appropriate Wald test. P (P)<0.05 The value of (double tail) is considered significant.
Univariate and multivariate logistic regression analysis was used to evaluate the correlation between enterobacteriaceae and poor EI in the study cohort, and between poor EI end and 90 day functional end. All variables showed a relevant trend (P < 0.20) in the univariate analysis (poor EI outcome model: past history of antiplatelet use, history of stroke, blood glucose, UA, HCY, total cholesterol [ TC ], high density lipoprotein [ HDL ] ], low density lipoprotein [ LDL ] and Enterobacteriaceae, optimal outcome model: NIHSS score, enterobacteriaceae and poor EI, good functional outcome model: NIHSS score, enterobacteriaceae and poor EI). The predicted performance of poor EI and 90 day functional outcomes was assessed by comparing subject operating characteristics (ROC) curves using the multivariate model described above. The relative risk is expressed as a Odds Ratio (OR) with 95% Confidence Interval (CI).
Results:
for an overall summary of the features of study cohorts and validation cohort patients see table 2:
TABLE 2
Unless otherwise indicated, the data are median (interquartile range, IQR). Percentages are shown in brackets.
There was no significant difference in conventional parameters between EI and the bad EI group (pore EI):
a total of 36 moderately severe acute ischemic stroke patients were included in the modeling cohort to compare the flora between the EI and the poor EI group. After 7 days of treatment, 15 patients (41.7%) showed NIHSS improvement of > 40%, categorized as EI group, while the remaining 21 (58.3%) were categorized as bad EI group. Age, NIHSS score at admission, history of HBP or DM, blood glucose, etc. report potential indicators related to acute stroke prognosis, none of which showed significant differences between EI and adverse EI groups in this study, as shown in Table 3.
TABLE 3 Table 3
Unless otherwise indicated, the data are median (interquartile range, IQR). Percentages are shown in brackets.
There was a significant difference in the intestinal flora of the two groups, where the poor EI group was enriched in enterobacteriaceae:
principal coordinate analysis (PCoA) was used to determine if there were statistically differences in the flora structure of the two groups. For the unweighted UniFrac distances, the two groups were significantly different (Adonis test, r2=0.034, p=0.044) (fig. 1A), suggesting a difference in intestinal flora characteristics between the groups. We performed similar analyses using other distances, including weighting 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 indicate that early acute stage gut flora may be useful in distinguishing between EI and poor EI outcomes in stroke patients.
The taxonomic differences between the two groups were then assessed using LEfSe (a high-dimensional biomarker discovery algorithm). In LEfSe, higher LDA values indicate more significant differences in a certain species between test groups. In the experiments, it was observed that Enterobacteriaceae and Enterobacteriaceae (Enterobacteriaceae only) were significantly enriched in the poor EI group, while other taxa including actinomycetes, proteus A and actinomycetes were more enriched in the EI group (FIGS. 1B-C). Among them, the difference in enterobacteriaceae was most remarkable, and its LDA value was higher than 4 (fig. 1C). We further used the Mann-Whitney test to examine the significance of the relative abundance of enterobacteriaceae between the two groups. The enterobacteriaceae relative abundance was significantly increased in the poor EI group (median [ quartile range, IQR ]12.2% (2.8-29.0) versus 4.7% (1.1-7.9); mann-Whitney assay, P=0.027), almost 3 times that of the EI group (FIG. 1D). There were significant differences in Mann-Whitney test results for other taxa identified with LEfSe. However, the absolute abundance of other taxa is far lower than that of enterobacteriaceae.
Enterobacteriaceae predicts poor EI:
univariate and multivariate analysis were used to test whether enterobacteriaceae could be used to predict poor EI outcomes. The variables entered in the multi-factor logistic regression model are as follows: gender, age, smoking history, blood glucose, baseline NIHSS score, history of stroke, HBP, DM and HLP, which were previously reported potential confounding factors associated with acute stroke, and in addition, some laboratory assay values were added to the model, and single and multi-factor logistic regression analysis predicted poor EI outcomes for stroke patients in the study group, see 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) was associated with poor EI results. Fasting blood glucose, history of antiplatelet drug use, history of stroke, UA and HCY showed a trend associated with poor EI (P < 0.2). In the multivariate analysis adjusted for the variables described above, only Enterobacteriaceae (adjustment OR:6.27,95% CI: 1.16-33.73) still independently predicted poor EI outcomes. The predictive model contained enterobacteriaceae and fasting blood glucose, while UA, HCY, TC, HDL, LDL, history of stroke and history of antiplatelet drugs were removed because there was a multiple collinearity correlation between these variables. ROC plots of Logistic regression analysis showed that the predicted performance of this model (enterobacteriaceae + fasting glycemia) for poor EI outcome was 78.8% (area under the curve [ AUC ] estimated to be 78.8%), while enterobacteriaceae had similar efficiency (AUC estimated, 76.9%) for poor EI outcome (fig. 3A).
TABLE 4 Table 4
AUC represents area under the curve; anti-platelet drug history, anti-platelet drug prior to admission; HBP, hypertension; DM, diabetes; HLP, hyperlipidemia; TC, total cholesterol; TG, triglycerides; HDL, high density lipoprotein; 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 pressure; PBP, pulse pressure. * P <0.05. All the above values in the adjustment table are# adjusted.
Verification of enterobacteriaceae predicted defective EI:
in addition, a new clinical cohort was re-established from 2016-2017, containing a validated cohort of 88 patients (EI group: n=37 [42.0% ], bad EI group: n=51 [58.0% ]).
The age, sex, HBP, DM and NIHSS scores were similar for EI and bad EI groups at admission (see table 5 for analysis of bad factors related to EI between validation cohorts). Similar to the study group, the results of the LEfSe analysis showed that the enriched enterobacteriaceae was always the most important differential bacteria in the poor EI group compared to the EI group (fig. 2B-C). In the Mann-Whitney test, a significant increase in enterobacteriaceae was observed in the poor EI group compared to 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) FIG. 2D). ROC results showed that the area under the curve of enterobacteriaceae at poor EI outcome was 70.2%, whereas the predictive model of enterobacteriaceae plus fasting glycemia was 73.3% (fig. 3B).
TABLE 5
Unless otherwise indicated, the data are median (interquartile range, IQR). Percentages are shown in brackets.
Poor EI and enterobacteriaceae were used to predict 90 day functional outcome:
univariate analysis of 90 day long-term functional outcomes of ischemic stroke showed that baseline NIHSS scores, in the modeled cohort, enterobacteriaceae and poor EI were significantly correlated with 90 day best outcome (mRS 0-1) and good outcome (mRS 0-2) (both P < 0.05), for univariate and multivariate logistic regression analysis for predicting 90 day functional outcome for study cohorts patients, see table 5. Prediction of ischemic stroke 90 day functional outcome by corrected poor EI outcome for validation and whole queue As shown in Table 7, poor EI (OR: 0.059, 95% CI: 0.005-0.656) and NIHSS scores (OR: 0.287, 95% CI: 0.097-0.846) were able to independently predict 90 day best outcome (mRS 0-1) in a multivariate analysis adjusted for the variables and related confounding factors described above. We removed the Enterobacteriaceae family from the model because it correlated linearly with the poor EI. Likewise, poor EI outcomes (OR: 0.005, 95% CI: 0-0.392) and NIHSS scores (OR: 0.372, 95% CI: 0.179-0.770) are independent predictors of good outcomes over 90 days (mRS 0-2).
Adding poor EI outcomes to the model predicting 90 days of best and good outcomes 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) (fig. 4A).
In the validation queue, poor EI remains an independent predictor of 90 day best and good outcomes. Addition of poor EI results, which were confirmed in the validation queue, increased the best and good results for 90 days (AUC estimates increased from 0.747 to 0.802[ p=0.006 ] and 0.802 to 0.839[ p=0.016 ], respectively) predicted performance (fig. 4B).
The Enterobacteriaceae family is an independent predictor of 90-day best outcomes (OR: 0.249,95% CI: 0.089-0.693) and good outcomes (OR: 0.320,95% CI: 0.122) -0.844) throughout the large cohort (i.e., modeling+validation cohort). The addition of enterobacteriaceae also increased the predicted performance of the best and good outcomes for 90 days in the whole 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) (fig. 4C).
TABLE 6
* P <0.05. All the above values in the adjustment table are# adjusted.
TABLE 7
Multifactor logistic regression included age, gender, baseline NIHSS, HBP history, DM history, HLP history, CAD history, and smoking history. * P<0.05； ** P<0.01； *** P<0.001。
The entire queue, i.e., the sum of the modeled queue and the validated queue.
From the experimental results, the early acute stage intestinal enterobacteria after ischemic stroke are closely related to the 7-day poor EI outcome, and the traditional clinical variables predict that the efficacy of the 7-day poor EI outcome is lower. In addition, enterobacteriaceae in the early acute phase can become an independent predictor of 90-day functional outcome of ischemic stroke. The research result proves that the 7-day bad EI result is an independent predictor of 90-day functional outcome, and can improve the prediction performance of 90-day functional prognosis after ischemic cerebral apoplexy.
As mentioned above, EI has a positive correlation with the outcome of long term recovery, and improvement in neurological function is critical to clinical management, but not all patients can get satisfactory recovery through past standardized treatments. Identifying patients at high risk of poor EI is important for physicians to adjust treatment plans or develop new treatment methods. Indeed, EI is not a very precise term in clinic. Although the use of NIHSS scores is widespread, the "early" period is typically 2 hours to 30 days of onset time. In this study, baseline and day 7 NIHSS scores were used for evaluation. The median length of stay (LOHS) for acute stroke patients is typically 7 to 14 days, with seven-day follow-up being a point in time of an early recovery trend, being the maximum proportion of recovery per time period and being a predictive final outcome strength indicator. Patients on day 7 after the onset typically reach a relatively stable state, with almost half of the patients discharged around this time. Furthermore, the study showed that the value of this index in the acute stroke trial was superior to other typical endpoints (mRS scores at 30 and 90 days, NIHSS scores at 90 days). To assess early improvement or final functional outcome, demographic and clinical indicators including baseline NIHSS score, age, stroke severity, lesion site, past history of ischemic stroke, uric acid and diabetes have been demonstrated to be unstable in accuracy. An integrated age at admission and NIHSS score model correctly identifies 62.9% of patients who are not fully recovered or die, and 83.2% of patients who are fully recovered 100 days after acute stroke. However, the results of these indicators were not consistent as reported, and their efficacy for predicting early outcomes was poor in this study.
The inventors have found that the intestinal flora, in particular the 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 that significant differences in enterobacteriaceae between EI and bad EI groups and the effect of enterobacteriaceae on bad EI outcomes had reliable predictive performance (AUC 76.9% and 70.2%, respectively) in two independent queues. Negative results of traditional indicators may be due to heterogeneity in the ischemic stroke population, as most patients receive corresponding medication at the time of admission. In contrast, to date, there is no clinical treatment for the intestinal flora during hospitalization of patients with acute stroke. Study data further indicate that during patient hospitalization, its disturbed intestinal flora remains relatively stable. Changes in intestinal flora, including increases in enterobacteriaceae, are observed several days after cerebral stroke (1-4) and last for at least 3 weeks without significant changes. Thus, acute phase gut flora may be a reliable biomarker for outcome prediction.
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Sequence listing
<110> Zhujiang Hospital at university of south medical science
SOUTHERN HOSPITAL, SOUTHERN MEDICAL University
<120> use of enterobacteriaceae as ischemic stroke biomarker
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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ggactachvg ggtwtctaat 20
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<212> DNA
<213> Artificial sequence (Enterobacteriaceae)
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tacggagggt gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggtggtttgt 60
taagtcagat gtgaaatccc cgggctcaac ctgggaactg catctgatac tggcaagctt 120
gagtctcgta gaggggggta gaattccagg tgtagcggtg aaatgcgtag agatctggag 180
gaataccggt ggcaaggcgg ccccctggac gaagactgac gctcaggtgc gaaagcgtgg 240
ggagcaaaca gg 252
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.
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| CN111983160B (en) * | 2020-08-14 | 2022-08-09 | 中元伯瑞生物科技(珠海横琴)有限公司 | Application of nervonic acid in medicine for treating cerebral apoplexy |
| CN112553358A (en) * | 2020-09-19 | 2021-03-26 | 河北医科大学第二医院 | Application of microbial flora in cerebral apoplexy |
| CN112708686B (en) * | 2021-01-25 | 2022-10-21 | 温州医科大学慈溪生物医药研究院 | Application of intestinal flora in nerve injury detection |
| CN113215240A (en) * | 2021-04-14 | 2021-08-06 | 广东医科大学附属医院 | Method for researching relevance between unstable plaque cerebral infarction and intestinal flora, diagnostic marker and application thereof |
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