CN113201572A - Reagent for assisting diagnosis of tubercular meningitis - Google Patents

Abstract

The invention provides a screening method of a human group susceptible to tubercular meningitis or a risk assessment method for predicting pulmonary tuberculosis to develop into tubercular meningitis. Further screening for diagnosis of susceptibility to tubercular meningitis infection by detecting a fecal sample of the subject and detecting the relative abundance level of shigella coli.

Description

Reagent for assisting diagnosis of tubercular meningitis

Technical Field

The invention relates to the field of medical diagnosis, in particular to application of intestinal flora in disease diagnosis, especially in diagnosis of tubercle bacillus infectious diseases.

Background

Tuberculosis (TB) is an ancient infectious disease, is the cause of ten deaths worldwide and is also the leading cause of death from a single infectious disease. This disease usually affects the lungs (tuberculosis), but can affect other sites (extrapulmonary tuberculosis). Approximately 1000 million people are estimated to be infected with tuberculosis worldwide in 2019, approximately 120 million patients without HIV incorporated die due to tuberculosis, and approximately 20.8 million patients with HIV incorporated die from tuberculosis (Global tuberculosis report 2020.Geneva: World Health Organization; 2020. license: CC BY-NC-SA 3.0 IGO). Extrapulmonary tuberculosis is also an important component of tuberculosis, and Tuberculous Meningitis (TBM), the type of mortality and disability rate in tuberculosis, is important for humans to control tuberculosis (Marais, s.; Pepper, m.j.; Schutz, c.; Wilkinson, r.j.; mentjes, g.presence and outlet of Tuberculous Meningitis in a High HIV preference setting. plos ONE 2011; Sudha, k.; Rao, a.v.; Rao, s.n.; Rao, a.oxidative stress and infection in Tuberculous Meningitis. indian j. clinin biological m.2002,17, 34-41; Ray, g.; jejun s.115. CSF, balconis.120. in. environmental stress, 20. environmental stress, strain, emission. Tuberculous meningitis has various clinical symptoms, and the diagnosis needs cerebrospinal fluid smear, culture or Xpert positive (Suzaan Marais, Guy Thwaites, Johan F Schoeman. et al. Tuberculosis meninis: a uniform case definition for use in clinical research. Lancet Infect Dis 2010; 10: 803-12), and most Tuberculous meningitis are secondary to pulmonary tuberculosis, and cause attention of doctors only after occurrence of nervous system symptoms, so the diagnosis also has certain hysteresis, is difficult to diagnose clinically and has more sequelae after long time of diagnosis and treatment, and seriously affects the life quality of patients. But the incidence of tubercular meningitis is also not low. Adult tubercular meningitis accounts for 2.9% -5.9% of systemic tuberculosis. Brazil concerning a study of extrapulmonary tuberculosis, with 57217 patients with extrapulmonary tuberculosis, tuberculous meningitis occurs in about 6%, with an estimated 10 million new cases occurring each year (Gomes, t.et al, Epidemiology of extrapulmonary tuberculosis in Brazil: a tuberculosis model, bmc infection, dis.14,9 (2014)). Tuberculous meningitis occurs in higher incidence in children, 3.9% of children under 5 years old, 2.2% of children between 5 and 9 years old, and 1.3% of children between 10 and 14 years old, according to german monitoring data (ducible T, Tolksdorf K, karaginis I, Hauer B, Brodhun B, Haas W, et al. the burden of extrapulmonary and meniginia tubericulosis: An involvement of national surveillance data, Germany,2002to 2009 ro eurveill 2013). Investigation revealed that approximately 43 children were diagnosed with TBM in one hospital in Greece for 20 years (Buonsensso D, Serranti D, Valentii P.management of Central neural system cyberculosis in children: Light and shade, Eur Rev Med Pharmacol Sci 2010; 14: 845-53). Epidemiological investigations in China on tuberculous meningitis found that almost half of 1212 children diagnosed with tuberculosis and receiving antitubercular therapy during 2002to 2010 had extrapulmonary tuberculosis, with about 39% TBM (Wu XR, Yin QQ, Jiao AX, Xu BP, Sun L, Jiao WW, et al. Based on the characteristics of high morbidity and mortality and hysteresis in diagnosis, the method has important practical significance in researching the pathogenesis of the tuberculous meningitis and finding a new auxiliary evaluation index capable of assisting in diagnosing the tuberculous meningitis.

With the progress of research, people are more and more aware of the important influence of microorganisms living in our intestinal tracts on human bodies. Scientists have also recognized the important role played by the intestinal flora in regulating inflammation, inherited metabolic disorders, endocrine, and the like. And now more and more scientists also propose the existence of the intestinal tract brain axis, and further research finds that the intestinal microbiome damages the immune function of tuberculosis patients by changing butyric acid and propionic acid products. And Lipopolysaccharide (LPS) is the major component of the cell wall of escherichia coli, also known as endotoxin, and is a trigger for several inflammatory or infectious reactions in macrophages and other cells with CD14 receptors. Indeed, bacterial endotoxin (LPS) binding to Toll-like receptors (TLRs) can stimulate the secretion of Nitric Oxide (NO) and inflammatory substances (cytokines) such as tumor necrosis factor- α, interleukin 1- β, interleukin-6 by monocytes and macrophages. The Nemec scholars found that after gavage of e.coli in mice, an early systemic immune response was stimulated, which was manifested by elevated plasma TNF- α and organ NO levels. Muhammad Aslam, Nafes Ahmad and other scholars research shows that: inflammatory cytokine TNF-a enhances the permeability of brain capillaries, and TNF-a can reduce the activity of the Cldn-5 promoter and the expression of mRNA in mouse brain-derived endothelial cells. And the Cldn-5 has important significance for maintaining the permeability of the blood brain screen as an important tight-link protein for forming the blood brain barrier. The study also found that disturbances in the intestinal flora affect the blood brain barrier permeability (Braniste V, Al-Asmakh M, Kowal C, et Al. the gut microbiota influence blood-brain barrier permability in mice [ J ]. Sci Transl Med,2014,6(263):263ra 158).

Based on the existing research, the research on the relationship between tubercular meningitis and intestinal flora is creatively carried out, and the invention is completed.

Disclosure of Invention

In one aspect, the invention provides a screening method of people susceptible to tubercular meningitis, which is used for judging people susceptible to tubercular meningitis in people by evaluating the abundance condition of intestinal flora of people. Further screening for diagnosis of susceptibility to tubercular meningitis infection by detecting a fecal sample of the subject and detecting the relative abundance level of shigella coli.

Further, the present invention provides a method for predicting risk assessment of tuberculosis development into tubercular meningitis by assessing the abundance of intestinal flora in tuberculosis patients.

In a second aspect, the invention provides a detection kit for screening people susceptible to tuberculous meningitis in people, which comprises a reagent for detecting the abundance of intestinal flora in people, and the people susceptible to tuberculous meningitis is determined according to the abundance of intestinal flora.

Furthermore, the invention provides a detection kit for predicting the risk of tuberculosis developing into tuberculous meningitis, which comprises a reagent for detecting the abundance of intestinal flora of a tuberculosis patient, and the risk degree of tuberculosis developing into tuberculous meningitis of the tuberculosis patient is determined according to the abundance condition of the intestinal flora.

In a third aspect, the invention provides an application of a detection reagent for detecting the abundance of intestinal flora in preparing a detection kit for detecting a person susceptible to the tuberculous meningitis in a screened population, and the person susceptible to the tuberculous meningitis is screened by detecting the abundance of the intestinal flora of a person.

Furthermore, the invention provides an application of a detection reagent for detecting the abundance of intestinal flora in preparing a detection kit for predicting the risk of tuberculosis developing into tuberculous meningitis, and the degree of the risk of tuberculosis developing into tuberculous meningitis is determined by detecting the abundance of intestinal flora of tuberculosis patients.

The method can assist in screening or predicting the risk degree of the patient suffering from the tuberculous meningitis by detecting the abundance of the specific intestinal flora, is simple, convenient and easy to implement, has high patient compliance, can shorten the diagnosis time of the tuberculous meningitis, and improves the diagnosis accuracy. Further provides opportunities for the treatment of patients with tubercular meningitis, and obviously reduces the incidence of sequelae of patients with tubercular meningitis.

Drawings

FIG. 1 analysis of intestinal flora diversity of three groups of people (A) ACE diversity; (B) good's Coverage diversity; (C) shannon diversity.

FIG. 2 is a stack of horizontal intestinal flora between groups

FIG. 3 is a histogram of the genus level difference between groups and statistical analysis

FIG. 4 illustrates an exemplary ROC curve for species

Detailed Description

The detection method of intestinal flora used in the present invention may be a method known in the art, for example, a method of analyzing flora DNA extracted from feces by a 16S sequencing method.

Alpha diversity refers to the diversity condition within a particular habitat or ecosystem, which can indicate the degree to which the habitat is isolated by species, and is usually calculated using two important parameters, species abundance (species condition) and species uniformity (distribution condition). The six major classes, Chao1, ACE, Shannon, observed _ species, Simpson and Good's Coverage, are commonly used for the presentation of the alpha diversity index. Wherein the Chao1, ACE index are mainly concerned with species abundance information of the sample; good's Coverage reflects the low abundance OTU Coverage of the sample; the observed _ scenes represents the detected OTU type condition; simpson and Shannon mainly comprehensively reflect the richness and uniformity of species.

Through ACE diversity analysis of the intestinal flora, it was found that: comparing species abundance of the healthy control group, the tubercular meningitis group and the tuberculosis group, finding that the control group is significantly different from the tubercular meningitis group, the control group is different from the tuberculosis group, and the tuberculosis group is not different from the tubercular meningitis group, namely the species abundance of the tuberculosis and the tubercular meningitis is obviously reduced compared with the healthy control group, and the difference has statistical significance. Good's Coverage diversity reflects the low abundance OTU Coverage of the samples, with no difference in low abundance OUT Coverage among three different populations.

Shannon diversity mainly and comprehensively reflects the abundance and uniformity of species, and compared with a tuberculous meningitis group, a healthy control group, a tuberculous meningitis group and a tuberculous group, the healthy control group has richer species diversity and more uniform species distribution.

The composition of intestinal flora is analyzed from different human flora levels, and the obvious difference exists among the three groups, and the abundance of the large intestine-shigella (can be higher or lower) in the tuberculous meningitis group is obviously improved.

Therefore, the obvious increase of the abundance level of the large intestine-Shigella is a remarkable characteristic of patients with tuberculous meningitis, people susceptible to tuberculous meningitis can be screened according to the abundance level of the large intestine-Shigella, and the possibility that the patients with tuberculosis are likely to develop into the patients with meningitis is reliably evaluated.

The invention is further illustrated and explained by the following examples.

Example 1: fecal sample collection for different groups of people

1. Determination of the diagnostic criteria for tuberculosis:

sputum smear positive tuberculosis: meets one of the following:

(1)2 specimen sputum smears are positive for acid-fast bacilli.

(2) Positive acid-fast staining of 1 part of sputum specimen and positive chest imaging with pulmonary tuberculosis

(3)1 part of sputum specimen smear is acid-fast bacillus positive and mycobacterium culture positive, and the strain is identified as mycobacterium tuberculosis complex.

Positive tuberculosis diagnosis by only mycobacteria isolation culture: and pulmonary tuberculosis breast imaging shows, at least 2 sputum specimen smears are negative, mycobacteria culture is positive, and the strain is identified as a mycobacterium tuberculosis complex.

Molecular biological examination positive tuberculosis diagnosis: has positive pulmonary tuberculosis imaging performance and mycobacterium tuberculosis nucleic acid detection.

2. Diagnostic criteria for tubercular meningitis: HIV negative, older than 13 years. Definite or suspected tubercular meningitis

A) Clinical criteria are as follows: meningeal inflammatory conditions include fever, headache, vomiting for more than 2 weeks.

B) The support standard is as follows: i) the number of cerebrospinal fluid cells is more than or equal to 20/ul, mainly lymphocytes and the protein content is more than 2 g/L. Ii) CT scan showing exudate, infarct focus, hydrocephalus and various tuberculomas combined with iii) evidence of extracentral tuberculosis iv) response to antitubercular therapy

C) Exclusion criteria malaria, septic, fungal and cancerous meningitis.

Determination of diagnosis of tubercular meningitis: the kit is in accordance with A and C plus cerebrospinal fluid smear or cultured Mycobacterium tuberculosis PCR positive or ELISA IgM positive or acid-fast bacillus positive. Suggesting tuberculous meningitis: agreement with a and C and agreement with 3 or more evidences in B (6).

3. The judgment standard of healthy people is as follows: the past has no special history of lung diseases, routine physical examination blood routine, liver and kidney functions, electrolytes, cardiac enzyme, electrocardiogram, chest X-ray, ultrasound and other examinations have no abnormity.

4. Exclusion criteria

1) The antituberculosis treatment is carried out through systematic diagnosis and treatment.

2) And 6 months recently, the treatment with antibiotics is largely used for more than 1 week.

3) And gastrointestinal tract diseases such as Crohn's disease and ulcerative colitis.

4) And people with immune system suppression.

Collecting 5g of pollution-free excrement from morning of tuberculosis patients, tuberculous meningitis patients and healthy control groups into a sterile excrement collecting pipe, intercepting the inner part of the middle section of a sample by using a sterile toothpick or an excrement sampler, wherein the outer part is easy to pollute, part of bacterial DNA begins to degrade after contacting with air, and excrement collectors wash hands in the excrement collecting process, avoid secondary pollution in the collecting process and rapidly seal the collector after collection. Returning to the laboratory as soon as possible under the condition of low-temperature preservation, and storing in a refrigerator at-80 ℃ for later use.

Example 2: extracting DNA of the feces group (extracting DNA of the feces group by a kit method, the steps are as follows)

1. 0.25g of feces was added to the dried cylindrical tube. For stool samples with particularly high lipid, polysaccharide and protein content, a small amount of starting material may increase the uptake and purity of DNA.

2. 750ul of PowerBead solution was added to the dry cylindrical tube.

3. Add 60ul of solution C1, simple tumble several times or vortex.

4. The tube was heated at 65 ℃ for 10 minutes.

5. The cylindrical tube was vortexed horizontally using a vortexing adapter holder, vortexing at maximum speed for 10 minutes.

6. 13000g for 1 min.

7. The centrifuged supernatant was transferred to a new 2ml collection tube. Expected to be between 400-.

8. Add 250ul of solution C2 and vortex at maximum speed. Incubate at 2-8 ℃ for 5 minutes.

9. 13000g for 1 min.

10. To avoid collecting the pellet, 600ul of the supernatant was transferred to a new 2ml collection tube.

11. Add 200ul of solution C3 and vortex briefly.

12. 13000g for 1 min.

13. To avoid collecting the pellet, 600ul of the supernatant was transferred to a new 2ml collection tube. Avoid exceeding 750 ul.

14. 1200ul of solution C4 was added to the supernatant and vortexed for 5 seconds.

15. 650ul of the supernatant was added each time to the MB collection column, and 13000g was centrifuged for 1 minute. The filtrate was discarded until all supernatant was filtered through the collection column.

16. 500ul of solution C5 was added and 13000g was centrifuged for 1 minute.

17. The filtrate was discarded, and 13000g was centrifuged again for 1 minute.

18. Care was taken to move the MB column into a new 2ml collection tube.

19. Add 100ul of solution C6 to the center of the white filter.

20. 13000g were centrifuged for 1 min and the filter basket discarded. The DNA was stored in the filtrate.

Example 3: primer sequence for analyzing composition (B) of intestinal flora by 16S sequencing of fecal DNA

1. The experimental method comprises the following steps:

(1) after extracting genomic DNA from the sample, a conserved region of rDNA was amplified using specific primers with barcode (the sequence information of the primers is shown in Table 1)

Table 1: primer sequences

(2) The PCR amplification product was then recovered by cutting the gel and quantified using a QuantiFluor (TM) fluorimeter. And (3) mixing the purified amplification products in equal quantity, connecting a sequencing joint, constructing a sequencing library, and performing machine sequencing on Illumina PE 250.

2. Results of the experiment

(1) General analysis of three groups of people:

the general situation was analyzed and found (table 2): the healthy control group, the tubercular meningitis group and the tuberculosis group have no overall difference in age, sex and body quality index,

TABLE 2 demographic characteristics of HC, TBM, TB groups

(2) Three groups of population flora alpha diversity analysis

ACE diversity mainly concerns comparison among species abundance of samples in each group, ACE diversity index is selected for condition analysis among three groups, the average number of a healthy control group is 886.3, the average number of a tuberculous meningitis group is 600.4, the average number of a tuberculous group is 643.8, difference exists among the three groups of species abundance, the two groups among the groups compare the species abundance, the control group and the tuberculous meningitis group have obvious difference, p is 0.0035, the control group and the tuberculous group have difference, p is 0.0052, and no difference exists between the tuberculous group and the tuberculous meningitis group, p is 0.887. Therefore, the species abundance in tuberculosis and tubercular meningitis is obviously reduced compared with that in a healthy control group, and the difference has statistical significance. (FIG. 1-A)

Good's Coverage diversity reflects low abundance OTU Coverage of the samples, with a median of 0.998704 in the healthy control group, 0.998699 in the tuberculous meningitis group, 0.998632 in the tuberculous group, 0.6641 for the three group comparison, and P values greater than 0.05 for each two group comparison. There was no difference in low abundance OUT coverage among three different populations. (FIG. 1-B)

Shannon diversity is mainly combined with species abundance and uniformity. The mean of healthy control group was 5.519, mean of tubercular meningitis was 2.728, mean of tuberculosis was 4.168, and the three group comparison P was < 0.0001. Three groups differ in species abundance and uniformity. And performing multiple comparisons of two groups, wherein the difference p between the control group and the tuberculous meningitis group is less than 0.0001, the difference between the control group and the tuberculous meningitis group is 0.0062. The tuberculosis group is different from the tuberculous meningitis group, and p is 0.0035. It can be seen that the healthy control group has more abundant species diversity and more uniform species distribution than the tubercular meningitis group, the tuberculosis group and the tuberculosis group. (FIG. 1-C)

(3) Analysis of horizontal composition of different ethnic groups

We then analyzed the composition of the intestinal flora at the level of three ethnic groups. Referring to fig. 2, the stacking chart of relative abundance of intestinal flora at genus level is shown, the species composition of each sample at genus classification level is counted, the stacking chart visually shows the species abundance variation of different samples at genus level, the species with abundance mean top10 in all samples are selected for detailed display, the rest species are uniformly classified into Other category, and the tags which cannot be annotated to the level are classified into Unclassfied category. It can be seen that the composition of the horizontal intestinal flora in the genus is obviously different in three groups, and the abundance of the large intestine-Shigella in the tuberculous meningitis group is obviously increased.

The composition of the horizontal intestinal flora in the groups is shown in figure 3. Analyzing the relative abundance of species at the genus level between the healthy control group and the tubercular meningitis group, we found that there were 38 genera that differed between the two groups. The 36 genera are more enriched in the healthy control group, and the analysis of the 10 th relative abundance in the healthy control group shows that the statistical significance of the 4 genera are mainly found, wherein the genera are Blautia, Bifidobacterium, Fusicatenibacter and Anaerostipes, and the P values of the genera are 0.000957028, 0.006314, 0.01092169 and 0.048461 respectively. The 2 genera were more enriched in tubercular meningitis compared to healthy controls, with one of the genera being less abundant in both groups, not statistically, and more abundant in the two groups being Escherichia-Shigella coli, which had a P value of 0.003088121. It can be seen that Blautia, Bifidobacterium, Fusicatenibacter and Anaerostipes in the tuberculous meningitis group are reduced in abundance, and Shigella genus is relatively enriched in large intestine.

The relative abundance differences between the genus levels of the tubercular meningitis group and the tuberculosis group were analyzed, and we found that there were differences between the two groups at 4 genus levels. Of these, 3 genera are more enriched in tuberculosis, namely bacteriodes, Blautia, and CAG-56, and their P values are 0.003269736, 0.017492571, and 0.031275541, respectively. Compared with the statistical difference of the tubercular group, 1 genus of tubercular meningitis group was enriched with Shigella Escherichia Shigella, which is large intestine, and P value was 0.013218208.

It can be seen that the large intestine-shigella of tubercular meningitis group is higher than that of healthy control and tuberculosis group, and the difference has statistical significance.

(4) ROC curve (Receiver Operating characterization) analysis can be used to evaluate the correctness of species as biomarker, i.e. whether the sample can be divided into 2 groups as expected, and the method comprehensively considers the sensitivity and accuracy of species discrimination capability. By using the R language pROC package and defaulting to carry out ROC curve analysis on species one by one according to a T test, a rank test and a remarkably different species union set between two groups obtained by the test, we find that the evaluation by selecting shigella coli as a biological marker has high specificity and sensitivity. For example, as shown in fig. 4, when the change of the abundance of the large intestine-shigella is used as the result of distinguishing the healthy population from the tuberculous meningitis population, the area under the curve is 0.901, and the AUC is predicted to have higher accuracy above 0.9. The optimal critical value is that when the intestinal tract large intestine-shiga abundance reaches 15.05%, the diagnosis specificity is 1 and the sensitivity is 0.778. Therefore, the method has high prediction and diagnosis values.

Claims (6)

1. A detection kit for screening out a person susceptible to tubercular meningitis in a population is characterized by comprising a reagent for detecting the abundance of intestinal flora in the population.

2. The kit of claim 1, wherein the abundance of gut flora is of the genus shigella.

3. A test kit for predicting the risk of tuberculosis developing into tuberculous meningitis comprises a reagent for detecting the abundance of intestinal flora of a patient with tuberculosis.

4. The kit of claim 2, wherein the abundance of gut flora is of the genus shigella.

5. An application of a detection reagent for detecting the abundance of intestinal flora in preparing a detection kit for detecting people susceptible to tuberculous meningitis in people, and the people susceptible to tuberculous meningitis are screened by detecting the abundance of human intestinal flora.

6. The application of a detection reagent for detecting the abundance of intestinal flora in preparing a detection kit for predicting the risk of tuberculosis developing into tuberculous meningitis determines the risk degree of tuberculosis patients developing into tuberculous meningitis by detecting the abundance of intestinal flora of the tuberculosis patients.

Images