Mycobacterium tuberculosis, commonly known asMycobacterium tuberculosis(tube bacterium), which causestuberculosisThe pathogen of.In 1882, German bacteriologist Robert Koch[6](Robert Koch, 1843-1910) found and proved to be the pathogen of human tuberculosis, which can invade various organs of the body, but can causepulmonary tuberculosisSee most.Tuberculosis is an ancient disease, which is widely distributed around the world and is the first cause of death from bacterial infectious diseases.[1]
Mycobacterium tuberculosis is aerobic and grows slowly, but it has strong resistance to dryness, cold, acid, alkali, etc. Under direct sunlight, it takes 2 to 7 hours for the tubercle bacillus in sputum to be killed.[5]
M. tuberculosisMycobacterium tuberculosis(tuberculosis bacillus) is the pathogen of human tuberculosis.It is a kind of obligate aerobic bacteria with positive acid fast staining.No flagella, fimbriae, microcapsule but not formedSporeAnd its bacterial wall has neitherGram positive bacteriaOfPhosphoteichoic acidThere is no gram negative bacterialipopolysaccharide。German bacteriologist Robert Koch[6](Robert Koch, 1843-1910) was discovered and proved to be the pathogen of human tuberculosis in 1882.Tuberculosis caused by this bacterium infecting human body is an infectious disease that seriously affects human life and health. After centuries of struggle with it, it has gradually been controlled. However, in recent years, due to the influence of various factors, the disease has become increasingly serious.[1]
Research History
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In 1882, German bacteriologist Robert Koch[6](Robert Koch, 1843-1910) First discovered and proved that Mycobacterium tuberculosis is the pathogen of tuberculosis.The bacteria can invade all tissues and organs of the body, but the most common infection is pulmonary infection.Tuberculosis has seriously affected human health and life, and human beings have struggled with it for many centuries.In Europe from the 17th century to the 18th century, tuberculosis was called the "white plague". Almost 100% of Europeans were infected and 25% died.With the continuous development of anti tuberculosis drugs and the improvement of health and living conditions, the incidence and mortality of tuberculosis once dropped significantly.Since the 1990s, due toAIDSAnd drug-resistant strains of Mycobacterium tuberculosis, application of immunosuppressants, drug abuse, poverty andPopulation mobilityAnd other factors, the global epidemic of tuberculosis has suddenly worsened.Tuberculosis has become the most serious global health problem in the world, especially in developing countries.About 1/3 of the world's people are infected with Mycobacterium tuberculosis, 9 million new cases occur every year, and 2 million people die of the disease.[1]
Biological characteristics
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Morphology and dyeing
Tuberculous branchingbacillusIt is a slender and slightly curved bacterium with a size of 1~4X0.4 μ m.Mycobacterium bovis is thicker and shorter.MycobacteriumBacteria ofcell wallThe lipid content is high, accounting for about 60% of the dry weight, and a large amount of mycotic acid is surrounded byPeptidoglycanThe outside of the layer can affect the penetration of dyes.Ziehl - Neelsen is generally used for mycobacteriumAcid fast stainingAt 5%Carbolic acidFuhong can be dyed after heating, but it is not easy to decolorize with 3% hydrochloric acid ethanol.If addedMethylene blueRe staining, the mycobacterium is red, while other bacteria and substances in the background are blue.The fungus has no spore and flagella.[1]
Acid fast staining of Mycobacterium tuberculosis × 1000[1]
Mycobacterium tuberculosis passes penicillinCycloserineorlysozymeInduction can affect the synthesis of peptidoglycan in cell wall,isoniazidAffects the synthesis of mycotic acid,macrophageThe role of lysozyme after phagocytosis of Mycobacterium tuberculosis can destroy peptidoglycan, which can cause it to becomeL-form bacteria, granular or filamentous.Isoniazid affects the synthesis of mycotic acid, which can be negative for acid fast staining.This morphological pleomorphic staining is often seen in specimens infected with tuberculosis both inside and outside the lung.In clinical tuberculous cold abscess and sputum samples, even non acid resistant Gram positive particles, formerly called Much particles, can be seen.The particle is in the body orcell cultureIt can return to acid resistant bacteria, so it is also L-type bacteria.[2]
Culture characteristics
Specific oxygen demand.Optimum temperature37 ℃, no growth below 30 ℃.Mycobacterium tuberculosiscell wallOflipidThe content is high, affecting the absorption of nutrients, so the growth is slow.Tuberculosis bacteria grow vigorously under the conditions of 40%~50% oxygen, 5%~10% CO2, 36 ℃± 5 ℃ temperature, and the appropriate PH value of 6.8~7.2.In generalculture mediumIt takes 18-24 hours for each generation to split, and only 5 hours for each generation with rich nutrition.[1]
Mycobacterium tuberculosis colony Roche medium[1]
The first separation requires nutrient rich medium.Commonly used are Lowenstein JensenSolid medium, containing egg yolk, glycerin, potato, inorganic salt andMalachite greenEtc.Malachite green can inhibithybrid bacteriumGrowth, easy to separate and long-term culture.Egg yolk contains lipid growth factor, which can stimulate growth.According to the number of inoculated bacteria, it can be seen in 2-4 weekscolony of bacteriaGrowth.The colony is granular, nodular or cauliflower shaped,Milky whiteColor or beige, opaque.stayliquid cultureThe bacteria in the base may grow rapidly due to the large nutrient contact surface.Generally, it can grow in 1~2 weeks.The positive rate of liquid culture was several times higher than that of solid culture in clinical specimens.[1]
biochemical reaction
Mycobacterium tuberculosis does not ferment sugars.The difference with Mycobacterium bovis is that Mycobacterium tuberculosis can synthesize nicotinic acid and reduceNitrateHowever, Mycobacterium bovis cannot.heatEnzyme contact testTo distinguish Mycobacterium tuberculosis fromnontuberculosis mycobacteriaIt is of great significance.Most of mycobacterium tuberculosis were positive in enzyme contact test, but negative in heat enzyme test;Most of the two tests were positive for nontuberculous mycobacteria.The inspection method of heat contact enzyme test is to warm the concentrated bacterial suspension in 68 ℃ water bath for 20 minutes, and then addH2O2。Observe whether there are bubbles. Those with bubbles are positive.[1]
Resistance
Mycobacterium tuberculosisethanolSensitive, died in 70% ethanol for 2 minutes.In addition, cell lipids can prevent cell water loss, so they are particularly resistant to drying.It remains infectious for 8 to 10 days after adhering to dust, and can survive for 6 to 8 months in dry sputum.Mycobacterium tuberculosis is sensitive to damp heat and is killed when heated in liquid at 62~63 ℃ for 15 minutes or boiled.Mycobacterium tuberculosis is sensitive to ultraviolet light.Direct sunlight for several hours can be killed, and can be used for disinfection of clothes, books, etc. of tuberculosis patients.[3]
Mycobacterium tuberculosis is resistant to acid (3% HCl or 6% H2SO4) or alkali (4% NaOH) and will not be affected for 15 minutes.Available onIsolation cultureIt is used to treat the samples contaminated with mixed bacteria and the viscous substances in the digestive samples.Mycobacterium tuberculosis 1:13 000Malachite greenIt is resistant and can be inhibited when added to the culture mediumhybrid bacteriumGrowth.[1]
Variability
TuberculosisMycobacterium can form, colonyvirulence, immunogenicity and drug resistance.The BCG vaccine is Calmette and Guerin (1908)bileThe attenuated live vaccine strain obtained from the potato culture medium after 230 generations in 13 years is now widely used for vaccination.[1]
Pathogenicity
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Mycobacterium tuberculosis does not produceExotoxin。Its pathogenicity may be related to theHistiocyteInflammation caused by internal mass reproduction, the toxicity of cell components and metabolic substances, and the immune damage caused by the body to the cell components are related.[1]
Pathogenic substance
1. CapsuleThe main components of the capsule are polysaccharides, some lipids and proteins.Its effects on Mycobacterium tuberculosis include: ① The capsule can bind to complement receptor 3 (CR3) on the surface of phagocytes, which helps Mycobacterium tuberculosishost cellAdhesion and invasion on; ②CapsuleThere are many enzymes inmacromoleculeSubstance for the nutrition required by the reproduction of invasive Mycobacterium tuberculosis; ③The capsule can prevent the harmful substances of the host from entering Mycobacterium tuberculosis, even small molecule NaOH is not easy to enter.Therefore, when the tuberculosis specimen is digested with 4% NaOH, the general bacteria will be killed quickly, but the mycobacterium tuberculosis can tolerate for tens of minutes.The capsule can also inhibit the invasion of Mycobacterium tuberculosisPhagosomeAndlysosomeIntegration of.[1]
2. LipidsAccording to experimental research, the bacterial virulence may be related to the complex lipid components contained in it, especially glycolipids. ①Cord factorIt is a kind of glycolipid combined by mycotic acid and trehalose.It can make bacteriaLiquid mediumIt is arranged in a winding rope shape in the middle.This factor is closely related to the virulence of Mycobacterium tuberculosis.It can destroy cellsmitochondrial membrane, ImpactCellular respiration, suppressionwhite blood cellWandering and causing chronic granuloma.If it is extracted from bacteria, the bacteria will lose their virulence. ②Phospholipids: can promotemonocyteHyperplasia andmacrophageChange toEpithelioid cellsTo formTuberculous nodule。③sulphuric acidCerebroside(sulfatide): can inhibit the expression ofPhagosomeThe combination with lysosome enables Mycobacterium tuberculosis toPhagocyteMedium and long-term survival. ④Wax D: It is a complex of peptide glycolipid and mycotic acid, which can be extracted from toxic strains or BCG vaccine with methanol, and has adjuvant effect, which can stimulate the body to produceDelayed hypersensitivity。[1]
3. ProteinMycobacterium tuberculosis contains a lot of protein, which is related to the pathogenicity of bacteria as a virulence factor.Tuberculin is the main component of bacterial protein, which can cause hypersensitivity reaction in the body when combined with wax D, causingTissue necrosisAnd systemic poisoning symptoms, andTuberculous nodulePlay a certain role in.[1]
Disease caused
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Mycobacterium tuberculosis can invade susceptible organism through respiratory tract, digestive tract or skin damage, causing tuberculosis of various tissues and organs, among which, it is caused through respiratory tractpulmonary tuberculosisIs the most.Mycobacterium tuberculosis can be inhaled through droplets or dust containing bacteria, so pulmonary tuberculosis is more common.[1]
1. Intrapulmonary infection (1)Primary infection: It usually occurs in children.Most of the symptoms are low fever, night sweats, and a small amount of hemoptysis.There are a lot of alveolimacrophageA few live Mycobacterium tuberculosis enter the alveoli and are engulfed by macrophages.Because the bacterium has a large amount of lipid, it can resistlysozymeThe continuous reproduction will destroy the macrophages, and a large number of bacteria released will cause inflammation in the alveoli, which is called the primary focus.Primary infection due to lack of organismSpecific immunity, Mycobacterium tuberculosis often reaches the hilum of lung through lymphatic vesselslymph gland, causing hilum of lungLymph node enlargementAnd causeLymphangitis, the three are calledPrimary syndrome。A few of the patients may be cured by blood andlymphatic system, spread to bone, joint, kidney, meninges and other parts to cause corresponding tuberculosis.More than 90%Primary infectionFormation of fibrosis or calcification,Heal without cureHowever, there is still a certain amount of Mycobacterium tuberculosis latent in the focus for a long time.[4]
⑵ Secondary infection: This type is the most common type in adults.Pulmonary lesions were also common.Most of them were caused by bacteria from the primary latent focus, and a few were caused by exogenous bacteria.The main symptoms are low fever, night sweats, hemoptysis, etc.It has long been a chronic disease, with a few acute attacks.Due to the difference in immunity and treatment, there are many different types of lesions.The focus of secondary tuberculosis tends to limit, but the discharge of bacteria is more important in epidemiology.[4]
2. Extrapulmonary infection In some patients, Mycobacterium tuberculosis may enter the blood circulation and cause intrapulmonary and extrapulmonary spread, such as brain and kidney tuberculosis, and sputum bacteria ingested into the digestive tract may also cause intestinal tuberculosisTuberculous peritonitisEtc.[4]
Immunity
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Immune mechanism
Mycobacterium tuberculosis is a facultative intracellular parasite, whose immunity is mainly T cell based cellular immunity, and CD4 T cell is the mainImmune cell。Although the body can produce antibodies against Mycobacterium tuberculosis, this antibody has no protective effect.Release after activation of CD4T cellscell factorstimulatemacrophageMacrophages kill and clear Mycobacterium tuberculosis, so that the focus inflammation disappears and the body recovers.The clearance of the focus is related to the size of the focus. When the focus is too large and forms a fibrous capsule, macrophages cannot enter to kill bacteria, and bacteria will enter a resting state in the focus, resulting in latent infection.[1]
Tuberculosis immunity belongs to infection immunity, also known asBactericidal immunityThat is, only when Mycobacterium tuberculosis or its components exist in the body can it have immunity.Once all the mycobacterium tuberculosis or its components in the body disappear, immunity also does not exist.[1]
hypersensitivity
With the protection of the body against Mycobacterium tuberculosis, there can also be delayed type hypersensitivity, both of which are mediated by T cells.Hypersensitivity is related to both immune mechanism and pathogenic mechanism of bacteria.[1]
PPD testIt is a skin test using tuberculin to determine whether the body can cause hypersensitivity to Mycobacterium tuberculosis.[1]
In the routine test, 5 units of 2 PPDs were injected into the skin of both forearms respectively. After 48-72 hours, those with redness, swelling and induration exceeding 5 mm were positive, and those with ≥ 15 mm were strong positiveclinical diagnosismake sense.If the redness and swelling on the PPD-C side is greater than that on the BCG-PPD side, it is infection.On the contrary, the BCG-PPD side is larger than the PPD-C side, which may be caused by BCG vaccination.[1]
The negative reaction indicates that the patient has not been infected with Mycobacterium tuberculosis, but the following conditions should be considered: ① In the initial stage of infection, it takes more than 4 weeks for the patient to develop hypersensitivity due to Mycobacterium tuberculosis infection; ②The elderly; ③Patients with severe tuberculosis or suffering from other infectious diseases, such as low cellular immunity caused by measles; ④Acquired cellular immunity is low, such as AIDS or cancerImmunosuppressantThe.In order to eliminate false negative, some domestic units add asepticPhytohemagglutinin(PHA) injection, 0.1ml contains 10 μ g for skin test.If the 24h redness and swelling are greater than the PHA pimple, the cellular immunity is normal; if there is no response or the response does not exceed the PHA pimple, the immunity is low.[1]
Tuberculosis gamma interferon release test
T-cell interaction gamma release assays (TIGRA, also known as IFNGRA or GRA) have been adopted in recent yearsELISA(ELISA) orEnzyme-linked immunospot(ELISPOT) The whole blood orPeripheral blood mononuclear cellsThe IFN - γ test release reaction of specific antigen of Mycobacterium tuberculosis is used for the diagnosis of latent infection of tuberculosis.IFN - γ isTh1 cellsSecretorycell factorIt can not only reflect the Th1 cell immunity of tuberculosis in the body, but also closely related to the antigen content of tuberculosis in the body.T cells sensitized by Mycobacterium tuberculosis antigen can produce high levels of IFN - γ when encountering similar antigens, so they are used for the diagnosis of latent infection of tuberculosis.The specificity is stronger than PPD.[1]
Direct smear microscopy
Specimen directsmearOr smear after bacteria collection and stain with acid fast.If acid fast positive bacteria are found, a preliminary diagnosis can be made.Ziehl Nielsen method is generally used for acid fast staining.In order to enhance staining, IK (intensified Kinyoung) method can be used.Staining with carbolic acid fuchsin overnight and decoloring with 0.5% hydrochloric acid ethanol for 30s, most L-forms of Mycobacterium tuberculosis can also be stained.To improve the sensitivity of microscopic examination, gold amine staining can also be usedfluorescence microscopeMycobacterium tuberculosis showed golden yellow fluorescence.[1]
Isolation culture
Inoculate the neutralized bacteria collection materials into the solid culture medium, add rubber stoppers at 37 ℃ at the mouth of the container, and observe once a week.Mycobacterium tuberculosis grows slowly, and it usually takes 2 to 4 weeks to become visible to the naked eye.For liquid culture, the bacteria collection material can be dropped into the culture medium containing serum, and the particles can grow at the bottom of the tube in 1~2 weeks.The sediment can be taken as a smear, which can quickly obtain results, and further determine and distinguish between Mycobacterium tuberculosis andnontuberculosis mycobacteria。Domestic scholars have proved that Mycobacterium tuberculosis L-form can exist inblood corpuscleInside or adhering tocell surface。Such patients tend to have faster ESR, and inoculation of hypertonic Mycobacterium tuberculosis L-type culture medium immediately after hemolysis with hypotonic saline can improve the positive rate of culture.[1]
Animal testing
Inject the collected material subcutaneously into the groin of guinea pigs, and ifLymph node enlargement，Tuberculin testYang turns, and then dissection can be carried out.Observe whether the lung, liver, lymph nodes and other organs have tuberculous lesions, and conduct morphology, culture and other examinations.If no disease is found after 6-8 weeks, anatomical examination should also be carried out.[1]
rapid diagnosis
Generally, 5x10 bacteria are required for smear inspection3~4/Ml, culture needs 1x10two/Ml, when the number of bacteria in the sample is less than this number, it is not easy to obtain positive results, and the culture takes a long time.applicationPolymerasechain reaction(PCR) amplification technology is applied to the identification of Mycobacterium tuberculosis DNA. Only a few bacteria are needed in each ml to obtain positive results, and the results are obtained 1-2 days later.Pay attention to the pollution of experimental equipment during operation to avoid false positives.alsoBacterial L-formDue to wall deficiency and compensatory cell membrane thickeninglysozymeIt can not break the cell membrane to release DNA, resulting in false negative PCR.After the cells are broken by fully grinding with a tissue grinder, the positive reaction can occur.Conditional units use BACTEC method, using palmitic acid containing 14C as carbon sourcesubstrate7H12 medium, measure the amount of 14C produced in the process of bacterial metabolism to calculate whether there are acid fast bacteria in the sample, and report in 5-7 days.[1]
Prevention and control principle
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prevention
In the past 20 years, international organizations have proposed that the main methods to control tuberculosis include: finding and treating those with positive sputum bacteria to prevent and control the source of infection.BCG vaccine(BCG) vaccination.At present, the common view is that vaccination is not enough to prevent infection, but it can significantly reduce the incidence and severity of newborns.Dots strategy is a strategy that WHO has upgraded to ensure the success of tuberculosis control strategy based on the experience of 20 years.[4]
treatment
At present, drug chemotherapy is the main treatment for tuberculosis.Li Fuping、isoniazid、Ethambutol、streptomycin，PyrazinamideThey are first-line anti tuberculosis drugs.Surgical treatment can be taken for drug failure or life-threatening localized lesions.There are also symptomatic treatments that are important in the treatment, such as the treatment of fever and the prevention and treatment of hemoptysis.[4]
