(1) primary infection: mostly occurs in children. There are a large number of macrophages in the alveolar minority living Mycobacterium tuberculosis into the alveoli is, macrophage phagocytosis. Since there are a large number of bacteria lipid resistant lysozyme continue to multiply, the release of a large number of bacteria in the alveolar macrophage destruction caused by inflammation, known as the primary tumor. The initial infection of the body due to the lack of specific immunity, Mycobacterium tuberculosis often via lymphatics reach the hilar lymph nodes, causing hilar lymphadenopathy, said primary syndrome. At this point, may have a small amount of M. tuberculosis into the blood, spread to the body, but not necessarily obvious symptoms of (called recessive bacteremia); at the same time within the lesion macrophage-specific antigen presented to the surrounding lymphocytes . Infection in 3 to 6 weeks, the body produces specific cellular immune also appear hypersensitivity. Lesions in tuberculous mycobacterial cell wall phospholipids, on the one hand, the stimulation of macrophages into epithelioid cells, the latter merging the formation of multinucleated giant cells (Langerhans giant cells) or mitotic, on the other hand inhibit protease dissolution of the organization, the lesion tissue does not dissolve completely, caseous necrosis, wrapped around the epithelioid cells outside lymphocytes, macrophages and fibroblasts, the formation of nodular tuberculosis (tuberculous granuloma) typical pathological features of tuberculosis. Approximately 5% of the infection may develop active tuberculosis, the small number of patients immunocompromised, menstrual blood and lymphatic system and spread to the bones, joints, kidneys, meninges and other parts of the cause TB. More than 90% of the original infection to the formation of fibrosis or calcification, and heal, but Mycobacterium tuberculosis is still a certain amount of long-term latent lesions often, not only to stimulate the body to produce an immune future endogenous infection origins.
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No. 2
Mycobacterium tuberculosis Mycobacterium tuberculosis (M.tuberculosis), commonly known as Mycobacterium tuberculosis, TB is caused by bacteria. May invade the body organs, but tuberculosis is the most common. Tuberculosis still remains an important infectious diseases. Estimated world population of 1/3 of Mycobacterium tuberculosis infection. According to WHO reports, about 800 million new cases each year occur, at least 300 people die from the disease. Before the founding of our mortality 200-300 / 100,000, ranking first in the death of a variety of diseases reason, after the founding of the people's living standards improve, improve health status, particularly to carry out mass prevention, children's universal BCG vaccination, tuberculosis greatly reduced morbidity and mortality. It should be noted that some parts of the world due to AIDS, drug abuse, use of immunosuppressive drugs, alcoholism and poverty and other reasons, the incidence of another rise. Biological traits Morphology and staining of Mycobacterium tuberculosis bacilli slender, slightly curved, size 1 ~ 4X0.4? M (Figure 14-1A). Mycobacterium bovis is relatively chunky. Bacteria of the genus Mycobacterium cell wall lipid content is high, about 60% by dry weight, in particular a large number of mycolic acids (mycolic acid) surrounds the outside of the peptidoglycan layer, can affect the penetration of the dye. Mycobacterium general use Zini (Ziehl-Neelsen) acid-fast staining, after a 5% carbolfuchsin warming staining can be infected, but with 3% hydrochloric acid, ethanol is not easy to bleaching. If coupled stained with methylene blue, the red mycobacteria, bacteria and other substances in the blue background. In recent years, found in the cell wall of Mycobacterium tuberculosis outer layer of the capsule there. Generally due to damaged and difficult to see when the producers. If in the preparation of electron microscopy specimens were fixed before treatment with gelatin to prevent dehydration capsular contraction. In the electron microscope, you can see the outer cell layer of thick transparent areas, namely capsule, capsule Mycobacterium tuberculosis have a protective effect. Mycobacterium tuberculosis in vivo foreign economic penicillin, cycloserine or lysozyme induction can affect the synthesis of peptidoglycan cell wall, isoniazid affect mycolic acid synthesis, macrophages engulf the role of Mycobacterium tuberculosis lysozyme can damage the peptidoglycan, which can lead to L-type, granular or filamentous (Figure 14-1B and C). Isoniazid affect mycolic acid synthesis, can become negative for acid-fast staining. This form of polymorphic staining varied specimens of tuberculosis infection in the lungs and outside often can be seen. Clinical tuberculous cold abscess and sputum specimens have even seen non-acid-resistant Gram-positive granules, formerly known as Much particles. The particles in vivo or in cell culture can be returned as acid-fast bacilli, hence the L-type. Obligate aerobic culture characteristics. The optimum temperature is 37? C, less than 30? C no growth. Higher lipid content of the cell wall of Mycobacterium tuberculosis, affect the absorption of nutrients, so slow growth. Every generation needs to split time in the general medium 18 ~ 24h, while nutritious just 5h. Mycobacterium tuberculosis colonies - Jensen medium initial separation requires nutrient-rich medium. Commonly used Roche (Lowenstein-Jensen) solid medium containing egg yolk, glycerol, potatoes, salt and malachite green and so on. Malachite green inhibits bacteria growth, ease of separation and long-term culture. Egg yolk lipid containing growth factors can stimulate growth. According to the number of inoculated bacteria, usually 2 to 4 weeks visible colony growth. Colonies were granular, nodular or cauliflower-shaped, white or beige, opaque. May be due to the large surface of contact with nutrition, more rapid growth of bacteria in a liquid medium. Generally 1 to 2 weeks to grow. Examination of clinical specimens in liquid culture than solid culture positive rate several times. Biochemical reactions Mycobacterium tuberculosis does not ferment sugars. M. bovis and M. tuberculosis is the difference between acid and nitrate reduction can be synthesized, and M. bovis can not. Hot catalase test for differences between M. tuberculosis and non-tuberculous mycobacteria is important. Most of Mycobacterium tuberculosis catalase test positive, catalase test was negative and heat; non-tuberculous mycobacteria are mostly two kinds of tests were positive. Hot catalase test method is to check the concentration of the bacterial suspension _set_ 68? C water bath heating 20min, then add H2O2. Observe whether bubbles, bubbles were positive. Mycobacterium tuberculosis resistant to lipid-containing cell wall, so the sensitivity of ethanol, 70% ethanol for 2min in death. Furthermore, the lipid prevents water loss cells, particularly the resistance of the dry strength. Adhesion to keep infectious 8 ~ 10d on the dust can survive in dry sputum 6 to 8 months. Mycobacterium tuberculosis heat sensitive, heat in the liquid 62 ~ 63? C 15min or be killed by boiling. UV-sensitive Mycobacterium tuberculosis. Direct sunlight for several hours can be killed, can be used in patients with tuberculosis clothes, books and other disinfection. Mycobacterium tuberculosis resistant to the presence of organic compounds in the environment is closely related, such as sputum can enhance the resistance of Mycobacterium tuberculosis. Because most disinfectants can sputum coagulation proteins, including bacteria around, the bacteria can not easily be killed. 5% carbolic acid in the absence of sputum 30min to kill Mycobacterium tuberculosis, sputum when needed 24h; 5% Lysol children 5min kill M. tuberculosis when no sputum, phlegm when needed 1 ~ 2h. Mycobacterium tuberculosis acid (3% HCl or 6% H2SO4) or base (4% NaOH) resistant, 15min unaffected. There can be used to deal with bacterial contamination of specimens and samples digested viscous substance when isolated and cultured. Mycobacterium tuberculosis resistant to 1:13 000 malachite green, plus inhibit bacteria growth in the medium. Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, cycloserine, ethambutol, kanamycin, sensitive amino salicylic acid, but long-term medication prone to drug resistance, and pyrazine amide resistance <5%. Variability of Mycobacterium tuberculosis can occur morphology, colony, virulence, immunogenicity and drug resistance mutation. BCG (BCG) is Calmette and Guerin 2 people (1908) Bovine tuberculosis after 13 years of 230 passages in medium containing glycerol, bile, obtained in potato live attenuated vaccine strains, are widely used to prevent vaccination. Mycobacterium refractory body containing a large amount due to lipids, representing 40% of the dry weight of TB maximum content of the cell wall, the outer wall of its hydrophobic lipid-rich, therefore, difficult to penetrate the general disinfectant, with the exception of large external conditions resistance to the failure of a conventional sterilization method is easy, 15% sulfuric acid or 15% sodium hydroxide solution for 30 minutes to kill the bacteria in general, but not kill M. tuberculosis. In the dark, dank place can survive for several months, can survive for hours under the sun exposure, -7 ℃ can survive in the following 4-5 years. But in boiling water (100 ℃) died a few minutes, so sterilized by boiling is the most effective and economical way. --- Alcoholic fatty alcohol solvent can penetrate its ester layer miraculous play, with 75% alcohol two minutes can kill it. Conditions of TB in 40% to 50% of an oxygen and a 5% ~ 10% CO2 and temperature of 36 ℃ ± 5 ℃, suitable PH value of 6.8 to 7.2 under the conditions of strong growth, and tuberculosis in the general media coli is grown, it must be grown on special media to serum, egg yolk, potatoes, and the glycerin contained some inorganic salts. So most vulnerable to violations of sufficient oxygen TB ends bloodstream, lungs and nutrient-rich bones. Mycobacterium tuberculosis, no matter what environment exists already shown, it is one of the finest family of bacterial strains. Lazy slow growth of Mycobacterium tuberculosis, no athletic ability, temperament lazy, its fastest pace of 18 hours proliferation generation, while most bacteria are breeding minutes or tens of minutes to generation, such as E. coli in about 20 minutes breeding a generation, 10 hours later, a reproduction of E. coli more than 1 billion, but a breed of Mycobacterium tuberculosis 18 hours to two. But do not underestimate or belittle the shape of Mycobacterium tuberculosis weary of this deceptive behavior with laziness, a large number of scientists in the laboratory or medical personnel in clinical practice with it for a long struggle repeatedly proven that means we do not struggle with it increased to a high level up. Mycobacterium tuberculosis prone to drug resistance. Commonly used in the solid medium containing isoniazid 1? G, streptomycin 10? G, rifampin 50? G can grow is resistant Mycobacterium tuberculosis. Resistant strains virulence weakened. Isoniazid can affect the synthesis of the cell wall mycolic acid-induced Mycobacterium tuberculosis to become L-type, this may be a cause of resistance to isoniazid. Drug susceptibility testing showed resistance to isoniazid, rifampicin and streptomycin while mostly still sensitive. Therefore, the treatment of many advocates isoniazid and rifampicin or pyrazinamide combination therapy to reduce drug resistance and enhance efficacy. Isoniazid-resistant strains of pathogenic also weakened clinically. Experiments show that guinea pigs infected with Mycobacterium tuberculosis often died at 6 weeks and saw a miliary liver lesions; whereas L-type infection tend to be the death of hundreds of talented, lack of typical tuberculosis lesions nodules. However, the characteristics of L-type a reply without a thorough treatment can lead to relapse. In recent years, the world of multi-resistant Mycobacterium tuberculosis strains gradually increased, and even cause outbreaks. Resistant Mycobacterium tuberculosis by spontaneous mutation (primary resistance) or by the inappropriate medication produced by mutant _select_ion (secondary resistance). The generation of multi-resistant but may be due to the latter. Resistance gene on the chromosome, the gene for resistance to different drugs is not connected, so the combination therapy effective. Isoniazid resistance and katG gene loss related. Susceptible strains of the gene, no resistant strains. Rifampicin major role in RNA polymerase. Gene encoding the enzyme (rpoB) mutations causing resistance to rifampin. 1999 China reported seven rifampin-resistant strains of the entire rpoB gene mutation. Sensitive strains not. Two pathogenic Mycobacterium tuberculosis do not produce exotoxin. Its pathogenic bacteria may multiply within cells caused by inflammation, immune cell composition and metabolic damage and the body of toxic substances produced by bacterial component related. Capsular pathogenic substances, lipids and proteins related. A main component of the capsule capsular polysaccharide, lipid and protein portion. Its effect on Mycobacterium tuberculosis are: ① capsule can 3 (CR3) combining the help of M. tuberculosis adhesion and invasion of host cells and phagocytosis of complement receptors on the cell surface; ② have capsular various enzymes can degrade host tissue macromolecules for M. tuberculosis invasion nutrients needed for reproduction; ③ capsule to prevent harmful substances into the host Mycobacterium tuberculosis, or small molecules such as NaOH is not easy to enter. The specimen tuberculosis digested with 4% NaOH, general bacteria quickly killed, but M. tuberculosis resistant to several tens of minutes. Mycobacterium tuberculosis after capsular invasion also inhibit fusion of phagosomes and lysosomes. 2. According to experimental studies of lipid may be related to bacterial virulence contained complex lipid composition, in particular to glycolipid more important. ① cord-like factor: is a mycolic acid and trehalose glycolipid binding. Bacteria can meander cord-like arrangement (Figure 14-2) was in the liquid medium. This factor is closely related to M. tuberculosis virulence. It can damage the mitochondrial membrane, cell respiration, inhibition of leukocyte migration and cause chronic granulomatous. If it made from the bacteria, the bacteria lost virulence. ② phospholipids: monocytogenes can promote and make inflammatory foci macrophages into epithelioid cells, thereby forming a tubercle. ③ cerebroside sulfate (sulfatide): inhibit phagocytic cells engulf combination with lysosomes that Mycobacterium tuberculosis can survive long-term in phagocytes. ④ waxy D: is a glycolipid and a peptide complex of mycolic acids, or from BCG strains have proposed as methanol, with an adjuvant action can stimulate the body to produce the delayed type hypersensitivity. 3 antigenic proteins, and wax D hypersensitivity occur upon binding to make the body, causing tissue necrosis and systemic symptoms, and play a role in the formation of nodules nodules. Disease caused by Mycobacterium tuberculosis can invade the respiratory, digestive or skin lesions susceptible organism, causing a variety of tissues and organs of tuberculosis, which causes tuberculosis through the respiratory tract most. Because there are a lot of normal intestinal flora sojourn, Mycobacterium tuberculosis must survive through competition and susceptible to cell adhesion. Alveolar no normal flora, Mycobacterium tuberculosis by droplets or droplet inhalation of dust bacteria, the TB is more common. 1 pulmonary infection due to the bacteria's virulence of the infection, the number of different immune status, tuberculosis can have the following two types of performance. (1) primary infection: mostly occurs in children. There are a large number of alveolar macrophages, a small number of live M. tuberculosis into the alveolar macrophage phagocytosis Jibei. Since there are a large number of lipid bacteria, resistant to lysozyme and continue to multiply, so that the destruction of macrophages, releasing a large number of bacteria cause inflammation in the lung, called a primary tumor. Initial infection due to lack of specific immune organism, Mycobacterium tuberculosis often arrive hilar lymph nodes via the lymphatic vessels, causing hilar lymph nodes, called primary syndrome. In this case, a small amount of M. tuberculosis into the bloodstream, spreading to the body, but not necessarily obvious symptoms (called recessive bacteremia); Meanwhile macrophages within the tumor-specific antigen presented to the peripheral lymphocyte . 3 to 6 weeks of infection, the body to produce specific immune cells, but also appears hypersensitivity. Cell wall of Mycobacterium tuberculosis lesions phospholipids, while stimulating macrophages into epithelioid cells, the latter or by nuclear fission and fusion to form multinucleated giant cells (ie, Langerhans giant cells), on the other hand inhibit proteases dissolution of the organization, so that the organization does not dissolve completely lesions produce caseous necrosis, epithelioid cells wrapped around the outside of lymphocytes, macrophages and fibroblasts, the TB nodules (ie TB granulomas) are typical pathological features of tuberculosis. About 5% after infection may develop active tuberculosis, including a small number of patients due to low immunity, can be blood and lymphatic system, spread to the bones, joints, kidneys, and other parts of the meninges caused corresponding tuberculosis. More than 90 percent of the primary infection formation of fibrosis or calcification, and heal, but the lesions often still a certain amount of long-term latent Mycobacterium tuberculosis, not only can stimulate the body to produce immunity can also become infected with the future of endogenous origin. (2) After the primary infection: lung lesions were also more common. Germs can be foreign (exogenous infection) or the original latent (endogenous infection) in the lesions. Because the body has specific cellular immunity, so after the primary infection is characterized by lesions confined, generally do not involve the nearby lymph nodes, caseous necrosis surrounded by cellulose can calcify and heal. If caseous nodules rupture, into the adjacent bronchus, you can release large amounts of the formation of voids and to Mycobacterium tuberculosis in sputum. 1990 foreign reports of various types of tuberculosis, sputum samples detected in 40% of L-type. It was noted that in recent years saw a lesion morphology atypical acid-fast bacilli, but no typical tuberculous nodules, called "non-reactive tuberculosis." L with Mycobacterium tuberculosis infection in experimental animals, but also saw a similar situation. This is due to the lack of Mycobacterium tuberculosis L-type cell wall lipid composition, can stimulate the formation of nodules, and only lymph nodes and caseous necrosis. Single judge from pathological changes, often mistaken for chronic lymphadenitis. Some of the 155 cases the diagnosis was made retrospective study of chronic lymphadenitis wax block specimens for immunization with BCG antibody staining, 68.9% positive, 60% of the acid-fast staining acid particles. Description of cases in large part related to L-type M. tuberculosis. Clinically, this should be noted, to prevent misdiagnosis and misdiagnosis. 2 extrapulmonary Mycobacterium tuberculosis infection in some patients may cause the lungs into the bloodstream, outside spread, such as the brain, kidney tuberculosis, sputum is swallowed into the digestive tract can also cause intestinal tuberculosis, tuberculous peritonitis. Abroad have reported 332 cases of blood samples cultured only two cases of Mycobacterium tuberculosis, but this subcutaneous injection of guinea pigs infected with TB specimens of 12%. M. tuberculosis described in the blood than most general bacteria spread type, but a type L Growth difficult. In recent years, many new reports of extrapulmonary tuberculosis, Mycobacterium tuberculosis bacteria detection rate than L-type: such as children 10 cases of tuberculous meningitis cerebrospinal fluid culture, nine cases of cultured L-type, only one case of bacterial type. Difficulty urinating senile prostatic hypertrophy, postoperative pathological acid-fast bacilli accounted for 61.2% of the L-type, no one was typical acid-fast bacilli. Conventional culture-negative chronic prostatitis, nearly 1/3 of the L-type detection of acid-fast bacilli. Infertile men semen checklist to see acid-fast bacilli 7% acid L-type single-see 14% of L-type electron microscope examination showed adsorbed on the sperm head and tail. L Mycobacterium tuberculosis infection in mice, between 73% saw Leydig inflammation L-type acid-fast bacilli. Third, immunity Immune mechanisms of intracellular Mycobacterium tuberculosis infection is bacteria, the immune T cells mainly based cellular immunity. T cells can not directly and intracellular bacteria, and the infected cells must first reaction, leading to the collapse of the cells, release of Mycobacterium tuberculosis. Although the organism Mycobacterium tuberculosis produce antibodies, but antibody only contacts with the release of bacteria from a supporting role. Mycobacterium tuberculosis invades the respiratory tract, due to the alveoli of 80% to 90% macrophages, 10% lymphocytes (T cells accounted for the majority); primary alveolar macrophages not activated antimicrobial activity is weak, can not prevent swallowed growth of Mycobacterium tuberculosis, anti-tuberculosis can be brought to his office. But presenting antigens, so that the surrounding T lymphocyte sensitization. Sensitized lymphocytes may produce a variety of lymphokines, such as IL-2, IL-6, INF-?, Their interaction with the TNF-α to kill M. tuberculosis lesions. Lymphokines in INF-? Is a major, there are a variety of cells to produce INF-?, Infiltration has for NK,? / ΔT and CD4 +, CD8 + α / βT cells. Some of these cells can directly kill target cells, and some produce lymphokine-activated macrophages that cause respiratory outbreaks phagocytosis strengthened, resulting in the generation of reactive oxygen and reactive nitrogen intermediary intermediary rather kill the germs. Body T cell by antigen receptor (TCR) can be divided into two kinds of different: one from the α chain and β chains, called α / βT cells (including CD4 or CD8 flag), another by a chain and δ? chains, called? / δT cells (CD4 or CD8 mostly no sign). Human and mouse peripheral blood of the former? 90%, which is <10%. Antimycobacterial immunity in these two kinds of T cells play an important role in both. In the early stages of infection α / βT cells have not yet raised the peak of Mycobacterium tuberculosis by? / ΔT cell control. After contact with Mycobacterium tuberculosis? / ΔT cells that proliferate. Healthy people by mycobacterial extract stimulated 7 ~ 10d, peripheral lymphocytes in? / ΔT cells can be increased, its role and α / βT cells also can kill Mycobacterium tuberculosis. In recent years proved that mice infected with Mycobacterium bovis after? / ΔT cells rapidly converge to the area of inflammation, proliferation, mainly V? 9δ2T cell sub_set_s, but people with active TB in this subgroup declined. Recently noticed? / ΔT is a cell-attack primarily the heat shock protein of M. tuberculosis (heat shock protein, HSP). HSP is a highly conserved protein in nature, both from prokaryotes to plants and animals, which have a 50% amino acid sequence homology. In normal organism little content, but infection, fever, and other malignant cells to ambient conditions to produce a large change i.e., also known as the stress proteins HSP (stress protein). HSP has many pathogens, the amino acid sequences have a common component, caused by different bacteria can cause subclinical immune to some extent the same, are considered non-specific immunity. TB infection are immune immune (infection immunity), there are bacteria, also known as immune, have immunity that only when the presence of Mycobacterium tuberculosis or a component when the body. Once the body or components of M. tuberculosis disappeared immune also will not exist. As the body's immunity and hypersensitivity Mycobacterium tuberculosis produce a protective effect, but also can see the generation of delayed-type hypersensitivity, the result both of which are mediated by T cells. You can see from the Koch phenomenon (Koch phenomenon), the M. tuberculosis initial subcutaneous injection of healthy guinea pigs, after 10 ~ 14d local unhealed ulcers, swollen lymph nodes near the bacteria spread to the whole body, the performance characteristics of primary infection. In terms of Mycobacterium tuberculosis to previously infected guinea pigs re-infected, within 1 ~ 2d partial quickly produce ulcers, easy to heal. Nearby lymph nodes enlargement, bacteria seldom spread of infection is characterized by the performance of the primary. When visible light reinfection ulcers, easy to heal, non-proliferation, indicating that the body has a certain immunity. However, re-infection occurs when ulcers faster, indicating that while there is an immune hypersensitivity to participate. In recent years, studies have shown that Mycobacterium tuberculosis induce different substances immunity and hypersensitivity. Tuberculin hypersensitivity is mainly caused by a common protein and waxy D, and immune from Mycobacterium tuberculosis ribosomal RNA (rRNA) cause. Two different antigen components activate different sub_set_s of T cells release a different lymphokine year. Tuberculin test tuberculin test tuberculin skin test is applied to determine whether the organism Mycobacterium tuberculosis can cause a hypersensitivity test. 1 tuberculin reagents in the past with the old tuberculin (old tuberculin, OT). Department of the Mycobacterium tuberculosis inoculated in glycerol broth, and incubated for 4 to 8 weeks after the heated filter made from concentrate. Diluted 2,000-fold with 5 units per 0.1ml. Currently using purified protein derivative (purified protein derivative, PPD). PPD has two kinds: one Mycobacterium tuberculosis BCG-PPD made PPD-C and BCG made. Each 0.1ml containing 5 units. 2 Test methods and significance of routine tests were taken two kinds of PPD 5 units injected within two forearm skin, swelling and induration 48 ~ 72h after more than 5mm as positive, ≥ 15mm is strongly positive, meaningful clinical diagnosis. If the PPD-C side swelling larger than BCG-PPD side of infection. Conversely, BCG-PPD PPD-C side is larger than the side, which is probably due to BCG vaccination. Negative reaction indicates uninfected Mycobacterium tuberculosis, but should consider the following: ① the initial infection, the need for more than 4 weeks to appear hypersensitivity due Mycobacterium tuberculosis infection; ② elderly; ③ or are suffering from severe tuberculosis patients Other diseases, such as measles immune cells resulting low; ④ get cell immunocompromised, such as AIDS or cancer and other immunosuppressive agents were used. To exclude false negative, and some domestic units plus sterile phytohemagglutinin (PHA) injection, 0.1ml containing 10μg skin test. If the swelling is greater than 24h PHA Picchu by normal cellular immunity, without response or no more than PHA Picchu by immunocompromised. Fourth, microbiological test method Signs and symptoms of TB are often not typical, though can take advantage of X-ray diagnosis, but the diagnosis still depends on the bacteriological examination. _Select_ specimens specimens according infection. Desirable sputum, bronchoalveolar lavage, urine, stool, cerebrospinal fluid, or chest, ascites. Other desirable extrapulmonary infection or blood or tissue cells secrete the corresponding parts. Direct smear smears or _set_s the bacteria smear with acid-fast staining. If the acid-positive bacteria can be found the initial diagnosis. Acid-fast staining is generally used Ziehl-Neelsen method. To enhance staining, the available IK (intensified Kinyoun) staining. The carbolfuchsin stained overnight with 0.5% hydrochloric acid in ethanol bleaching 30s, includes most of the L-type M. tuberculosis can also be colored. To improve the sensitivity of microscopic examination, can also be used auramine staining, fluorescence microscopy was significantly Mycobacterium gold fluorescence. Concentrated _set_ of bacteria strains after checking the first _set_, you can improve the detection rate. Culture and animal experiments must also be _set_ by the bacteria process to remove bacteria. Cerebrospinal fluid and chest, ascites no bacteria directly collecting bacteria can be precipitated by centrifugation. Sputum, bronchial lavage fluid, urine, fecal specimens subject to pollution 4% NaOH (sputum and the base 1:4, urine, bronchial lavage and the base 1:1) treatment 15min, long time easy to make the L-type M. tuberculosis deaths and non-tuberculous mycobacteria. Urine samples before adding 5% tannic acid, 5% acetic acid in each 0.5ml conical cylinder standing, taking sediment treatment. And then the treated material was pelleted by centrifugation. Take sediment smear staining. For further as culture or animal inoculation, should be neutralized with acid and then pelleted by centrifugation. Isolation and culture of the bacteria through the material and _set_ inoculated in solid medium, utensils, plus a rubber stopper at 37 ℃ oral culture, observed once a week. Slow growth of Mycobacterium tuberculosis, which normally takes 2-4 Zhou Changcheng visible off the bacteria. Liquid culture bacteria can be _set_ material dropping in serum-containing medium, you can in 1 to 2 weeks in the bottom of the tube and saw a particle growth. Take sediment smear, can quickly get results, and further for biochemical, and other measuring and differentiating susceptibility of Mycobacterium tuberculosis and non-tuberculous mycobacteria. Domestic scholars have proven Mycobacterium tuberculosis may be present in the L-cells in the blood on the cell surface or adhesion. Such patients often erythrocyte sedimentation rate, immediately after inoculation with hypotonic hemolysis hypertonic saline Mycobacterium tuberculosis L-type medium can improve the culture positive rate. Animal testing material after collecting bacteria inguinal subcutaneous injection in guinea pigs, 3 to 4 weeks after if the local lymph nodes, tuberculin test seroconversion can be dissected. Observed in lung, liver, lymph nodes and other organs or without tuberculosis, and for morphology, cultural and other tests. 6 to 8 weeks if still no disease, should also be dissected checked. Rapid diagnosis is not easy to obtain positive results in general smears bacteria need 5x103 ~ 4/ml, training needs 1x102/ml, the number of samples is less than this number when bacteria and cultures take longer. Currently has polymerase chain reaction (PCR) amplification technology for identification of Mycobacterium tuberculosis DNA, each containing only a few ml of bacteria can get positive, and 1? 2d outcome. Operation should pay attention to the pollution problem of experimental equipment, in order to avoid false positives. Also due to the lack of L-type bacteria and compensatory cell wall thickening, while not commonly used to make cell membranes rupture lysozyme release DNA, resulting in false-negative PCR. Fully with tissue grinders grind the cells rupture, there may be positive. BACTEC conditional units currently used method for containing carbon 14C palmitic acid substrate 7H12 medium, 14C in the measurement produced during the bacterial metabolism of the specimen is projected with acid fast bacilli, 5 ~ 7d can the report. In recent years, domestic and foreign clinical studies have shown that various types of tuberculosis patients in about 40% of the isolated L-type. After treatment of TB bacteria type disappears, L-often persist. There are holes in sputum of patients who have non-exclusive type of bacteria, may be detectable in about 8% of the L-shaped. Therefore, some scholars have suggested that L-type also detected several times as one of the criteria of TB activity, bacterial-type and L-type as sputum were negative to negative. Fifth, the principle of prevention Prevention of nearly 20 years of international organizations to control tuberculosis main methods are: ① detection and treatment of sputum-positive; ② neonatal BCG vaccination. Approximately 80% of the protected force. 1940s survey of part of the city of tuberculosis mortality 200/100 000 or more. After the liberation of improved sanitation, mortality from 1973 to 1977 display has been reduced to 30/100 000. But since 1979, three large-scale random checks nationwide epidemic declined very slowly. From 1979 to 1990 the annual prevalence rate of 2.8% descending, descending sputum-positive rate of 3.0%. Currently mortality 19/100 000, and the other is still two times communicable diseases. Ministry of Health requested in 2000 90% of neonatal BCG vaccination. According to the statistics vaccinated newborns who later than unvaccinated incidence decreased by approximately 80%. BCG is a live vaccine, vaccine directly affects the number of viable cells within the immune effect, so there are already freeze-dried vaccine supply. The new ribosomal RNA (rRNA) vaccine has been cause for concern, but still in the experimental stage. Treatment of rifampin, isoniazid, ethambutol, streptomycin for the first-line drugs. Rifampin and isoniazid may reduce the development of drug resistance. Severe infections can pyrazinamide and rifampin and isoniazid. 1g cheese kitchen or voids containing about 106 to 10 Mycobacterium tuberculosis. 1-6 per 105 may have one kind of bacteria resistant mutation, the need for two kinds of bacteria resistant to 1011, it is appropriate to the two drugs in combination.
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Biological traits
Morphology and staining of Mycobacterium tuberculosis bacilli slender, slightly curved, size 1 ~ 4X0.4? M (Figure 14-1A). Mycobacterium bovis is relatively chunky. Bacteria of the genus Mycobacterium cell wall lipid content is high, about 60% by dry weight, in particular a large number of mycolic acids (mycolic acid) surrounds the outside of the peptidoglycan layer, can affect the penetration of the dye. Mycobacterium general use Zini (Ziehl-Neelsen) acid-fast staining, after a 5% carbolfuchsin warming staining can be infected, but with 3% hydrochloric acid, ethanol is not easy to bleaching. If coupled stained with methylene blue, the red mycobacteria, bacteria and other substances in the blue background. In recent years, found in the cell wall of Mycobacterium tuberculosis outer layer of the capsule there. Generally due to damaged and difficult to see when the producers. If in the preparation of electron microscopy specimens were fixed before treatment with gelatin to prevent dehydration capsular contraction. In the electron microscope, you can see the outer cell layer of thick transparent areas, namely capsule, capsule Mycobacterium tuberculosis have a protective effect. Mycobacterium tuberculosis in vivo foreign economic penicillin, cycloserine or lysozyme induction can affect the synthesis of peptidoglycan cell wall, isoniazid affect mycolic acid synthesis, macrophages engulf the role of Mycobacterium tuberculosis lysozyme can damage the peptidoglycan, which can lead to L-type, granular or filamentous (Figure 14-1B and C). Isoniazid affect mycolic acid synthesis, can become negative for acid-fast staining. This form of polymorphic staining varied specimens of tuberculosis infection in the lungs and outside often can be seen. Clinical tuberculous cold abscess and sputum specimens have even seen non-acid-resistant Gram-positive granules, formerly known as Much particles. The particles in vivo or in cell culture can be returned as acid-fast bacilli, hence the L-type.
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Cultural characteristics
Obligate aerobic. The optimum temperature is 37? C, less than 30? C no growth. Higher lipid content of the cell wall of Mycobacterium tuberculosis, affect the absorption of nutrients, so slow growth. Every generation needs to split time in the general medium 18 ~ 24h, while nutritious just 5h. Mycobacterium tuberculosis colonies - Jensen medium initial separation requires nutrient-rich medium. Commonly used Roche (Lowenstein-Jensen) solid medium containing egg yolk, glycerol, potatoes, salt and malachite green and so on. Malachite green inhibits bacteria growth, ease of separation and long-term culture. Egg yolk lipid containing growth factors can stimulate growth. According to the number of inoculated bacteria, usually 2 to 4 weeks visible colony growth. Colonies were granular, nodular or cauliflower-shaped, white or beige, opaque. May be due to the large surface of contact with nutrition, more rapid growth of bacteria in a liquid medium. Generally 1 to 2 weeks to grow. Examination of clinical specimens in liquid culture than solid culture positive rate several times.
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Biochemical reactions
Mycobacterium tuberculosis does not ferment sugars. M. bovis and M. tuberculosis is the difference between acid and nitrate reduction can be synthesized, and M. bovis can not. Hot catalase test for differences between M. tuberculosis and non-tuberculous mycobacteria is important. Most of Mycobacterium tuberculosis catalase test positive, catalase test was negative and heat; non-tuberculous mycobacteria are mostly two kinds of tests were positive. Hot catalase test method is to check the concentration of the bacterial suspension _set_ 68? C water bath heating 20min, then add H2O2. Observe whether bubbles, bubbles were positive.
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Resistance
Containing Mycobacterium tuberculosis mycobacterial cell wall lipids under a scanning electron microscope, it is sensitive to ethanol, 70% ethanol for 2min in death. Furthermore, the lipid prevents water loss cells, particularly the resistance of the dry strength. Adhesion to keep infectious 8 ~ 10d on the dust can survive in dry sputum 6 to 8 months. Mycobacterium tuberculosis heat sensitive, heat in the liquid 62 ~ 63? C 15min or be killed by boiling. UV-sensitive Mycobacterium tuberculosis. Direct sunlight for several hours can be killed, can be used in patients with tuberculosis clothes, books and other disinfection. Mycobacterium tuberculosis resistant to the presence of organic compounds in the environment is closely related, such as sputum can enhance the resistance of Mycobacterium tuberculosis. Because most disinfectants can sputum coagulation proteins, including bacteria around, the bacteria can not easily be killed. 5% carbolic acid in the absence of sputum 30min to kill Mycobacterium tuberculosis, sputum when needed 24h; 5% Lysol children 5min kill M. tuberculosis when no sputum, phlegm when needed 1 ~ 2h. Mycobacterium tuberculosis acid (3% HCl or 6% H2SO4) or base (4% NaOH) resistant, 15min unaffected. There can be used to deal with bacterial contamination of specimens and samples digested viscous substance when isolated and cultured. Mycobacterium tuberculosis resistant to 1:13 000 malachite green, plus inhibit bacteria growth in the medium. Mycobacterium tuberculosis to streptomycin, isoniazid, rifampin, cycloserine, ethambutol, kanamycin, sensitive amino salicylic acid, but long-term medication prone to drug resistance, and pyrazine amide resistance <5%.
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Variability
Mycobacterium tuberculosis can occur morphology, colony, virulence, immunogenicity and drug resistance mutation. BCG (BCG) is Calmette and Guerin 2 people (1908) Bovine tuberculosis after 13 years of 230 passages in medium containing glycerol, bile, obtained in potato live attenuated vaccine strains, are widely used to prevent vaccination. Mycobacterium refractory body containing a large amount due to lipids, representing 40% of the dry weight of TB maximum content of the cell wall, the outer wall of its hydrophobic lipid-rich, therefore, difficult to penetrate the general disinfectant, with the exception of large external conditions resistance to the failure of a conventional sterilization method is easy, 15% sulfuric acid or 15% sodium hydroxide solution for 30 minutes to kill the bacteria in general, but not kill M. tuberculosis. In the dark, dank place can survive for several months, can survive for hours under the sun exposure, -7 ℃ can survive in the following 4-5 years. But in boiling water (100 ℃) died a few minutes, so sterilized by boiling is the most effective and economical way. --- Alcoholic fatty alcohol solvent can penetrate its ester layer miraculous play, with 75% alcohol two minutes can kill it.
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Conditional
TB in 40% to 50% of an oxygen and a 5% ~ 10% CO2 and temperature of 36 ℃ ± 5 ℃, suitable PH value of 6.8 to 7.2 under the conditions of strong growth, and the media in general is Mycobacterium tuberculosis no growth, it must be grown on special media to serum, egg yolk, potatoes, and the glycerin contained some inorganic salts. So most vulnerable to violations of sufficient oxygen TB ends bloodstream, lungs and nutrient-rich bones. Mycobacterium tuberculosis, no matter what environment exists already shown, it is one of the finest family of bacterial strains. Lazy slow growth of Mycobacterium tuberculosis, no athletic ability, temperament lazy, its fastest pace of 18 hours proliferation generation, while most bacteria are breeding minutes or tens of minutes to generation, such as E. coli in about 20 minutes breeding a generation, 10 hours later, a reproduction of E. coli more than 1 billion, but a breed of Mycobacterium tuberculosis 18 hours to two. But do not underestimate or belittle the shape of Mycobacterium tuberculosis weary of this deceptive behavior with laziness, a large number of scientists in the laboratory or medical personnel in clinical practice with it for a long struggle repeatedly proven that means we do not struggle with it increased to a high level up. Mycobacterium tuberculosis prone to drug resistance. Commonly used in the solid medium containing isoniazid 1? G, streptomycin 10? G, rifampin 50? G can grow is resistant Mycobacterium tuberculosis. Resistant strains virulence weakened. Isoniazid can affect the synthesis of the cell wall mycolic acid-induced Mycobacterium tuberculosis to become L-type, this may be a cause of resistance to isoniazid. Drug susceptibility testing showed resistance to isoniazid, rifampicin and streptomycin while mostly still sensitive. Therefore, the treatment of many advocates isoniazid and rifampicin or pyrazinamide combination therapy to reduce drug resistance and enhance efficacy. Isoniazid-resistant strains of pathogenic also weakened clinically. Experiments show that guinea pigs infected with Mycobacterium tuberculosis often died at 6 weeks and saw a miliary liver lesions; whereas L-type infection tend to be the death of hundreds of talented, lack of typical tuberculosis lesions nodules. However, the characteristics of L-type a reply without a thorough treatment can lead to relapse. In recent years, the world of multi-resistant Mycobacterium tuberculosis strains gradually increased, and even cause outbreaks. Resistant Mycobacterium tuberculosis by spontaneous mutation (primary resistance) or by the inappropriate medication produced by mutant _select_ion (secondary resistance). The generation of multi-resistant but may be due to the latter. Resistance gene on the chromosome, the gene for resistance to different drugs is not connected, so the combination therapy effective. Isoniazid resistance and katG gene loss related. Susceptible strains of the gene, no resistant strains. Rifampicin major role in RNA polymerase. Gene encoding the enzyme (rpoB) mutations causing resistance to rifampin. 1999 China reported seven rifampin-resistant strains of the entire rpoB gene mutation. Sensitive strains not.
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Pathogenicity
Mycobacterium tuberculosis do not produce exotoxin. Its pathogenic bacteria may multiply within cells caused by inflammation, immune cell composition and metabolic damage and the body of toxic substances produced by bacterial component related. Pathogenic substances The capsule, the lipid and protein related. A main component of the capsule capsular polysaccharide, lipid and protein portion. Its effect on Mycobacterium tuberculosis are: ① capsule can 3 (CR3) combining the help of M. tuberculosis adhesion and invasion of host cells and phagocytosis of complement receptors on the cell surface; ② have capsular various enzymes can degrade host tissue macromolecules for M. tuberculosis invasion nutrients needed for reproduction; ③ capsule to prevent harmful substances into the host Mycobacterium tuberculosis, or small molecules such as NaOH is not easy to enter. The specimen tuberculosis digested with 4% NaOH, general bacteria quickly killed, but M. tuberculosis resistant to several tens of minutes. Mycobacterium tuberculosis after capsular invasion also inhibit fusion of phagosomes and lysosomes. 2. According to experimental studies of lipid may be related to bacterial virulence contained complex lipid composition, in particular to glycolipid more important. ① cord-like factor: is a mycolic acid and trehalose glycolipid binding. Bacteria can meander cord-like arrangement (Figure 14-2) was in the liquid medium. This factor is closely related to M. tuberculosis virulence. It can damage the mitochondrial membrane, cell respiration, inhibition of leukocyte migration and cause chronic granulomatous. If it made from the bacteria, the bacteria lost virulence. ② phospholipids: monocytogenes can promote and make inflammatory foci macrophages into epithelioid cells, thereby forming a tubercle. ③ cerebroside sulfate (sulfatide): inhibit phagocytic cells engulf combination with lysosomes that Mycobacterium tuberculosis can survive long-term in phagocytes. ④ waxy D: is a glycolipid and a peptide complex of mycolic acids, or from BCG strains have proposed as methanol, with an adjuvant action can stimulate the body to produce the delayed type hypersensitivity. 3 antigenic proteins, and wax D hypersensitivity occur upon binding to make the body, causing tissue necrosis and systemic symptoms, and play a role in the formation of nodules nodules. Diseases caused by Mycobacterium tuberculosis can invade through the respiratory tract, gastrointestinal tract, or skin lesions susceptible organism, causing a variety of tissues and organs of tuberculosis, which causes tuberculosis through the respiratory tract most. Because there are a lot of normal intestinal flora sojourn, Mycobacterium tuberculosis must survive through competition and susceptible to cell adhesion. Alveolar no normal flora, Mycobacterium tuberculosis by droplets or droplet inhalation of dust bacteria, the TB is more common. 1 pulmonary infection due to the bacteria's virulence of the infection, the number of different immune status, tuberculosis can have the following two types of performance. (1) primary infection: mostly occurs in children. There are a large number of alveolar macrophages, a small number of live M. tuberculosis into the alveolar macrophage phagocytosis Jibei. Since there are a large number of lipid bacteria, resistant to lysozyme and continue to multiply, so that the destruction of macrophages, releasing a large number of bacteria cause inflammation in the lung, called a primary tumor. Initial infection due to lack of specific immune organism, Mycobacterium tuberculosis often arrive hilar lymph nodes via the lymphatic vessels, causing hilar lymph nodes, called primary syndrome. In this case, a small amount of M. tuberculosis into the bloodstream, spreading to the body, but not necessarily obvious symptoms (called recessive bacteremia); Meanwhile macrophages within the tumor-specific antigen presented to the peripheral lymphocyte . 3 to 6 weeks of infection, the body to produce specific immune cells, but also appears hypersensitivity. Cell wall of Mycobacterium tuberculosis lesions phospholipids, while stimulating macrophages into epithelioid cells, the latter or by nuclear fission and fusion to form multinucleated giant cells (ie, Langerhans giant cells), on the other hand inhibit proteases dissolution of the organization, so that the organization does not dissolve completely lesions produce caseous necrosis, epithelioid cells wrapped around the outside of lymphocytes, macrophages and fibroblasts, the TB nodules (ie TB granulomas) are typical pathological features of tuberculosis. About 5% after infection may develop active tuberculosis, including a small number of patients due to low immunity, can be blood and lymphatic system, spread to the bones, joints, kidneys, and other parts of the meninges caused corresponding tuberculosis. More than 90 percent of the primary infection formation of fibrosis or calcification, and heal, but the lesions often still a certain amount of long-term latent Mycobacterium tuberculosis, not only can stimulate the body to produce immunity can also become infected with the future of endogenous origin. (2) After the primary infection: lung lesions were also more common. Germs can be foreign (exogenous infection) or the original latent (endogenous infection) in the lesions. Because the body has specific cellular immunity, so after the primary infection is characterized by lesions confined, generally do not involve the nearby lymph nodes, caseous necrosis surrounded by cellulose can calcify and heal. If caseous nodules rupture, into the adjacent bronchus, you can release large amounts of the formation of voids and to Mycobacterium tuberculosis in sputum. 1990 foreign reports of various types of tuberculosis, sputum samples detected in 40% of L-type. It was noted that in recent years saw a lesion morphology atypical acid-fast bacilli, but no typical tuberculous nodules, called "non-reactive tuberculosis." L with Mycobacterium tuberculosis infection in experimental animals, but also saw a similar situation. This is due to the lack of Mycobacterium tuberculosis L-type cell wall lipid composition, can stimulate the formation of nodules, and only lymph nodes and caseous necrosis. Single judge from pathological changes, often mistaken for chronic lymphadenitis. Some of the 155 cases the diagnosis was made retrospective study of chronic lymphadenitis wax block specimens for immunization with BCG antibody staining, 68.9% positive, 60% of the acid-fast staining acid particles. Description of cases in large part related to L-type M. tuberculosis. Clinically, this should be noted, to prevent misdiagnosis and misdiagnosis. 2 extrapulmonary Mycobacterium tuberculosis infection in some patients may cause the lungs into the bloodstream, outside spread, such as the brain, kidney tuberculosis, sputum is swallowed into the digestive tract can also cause intestinal tuberculosis, tuberculous peritonitis. Abroad have reported 332 cases of blood samples cultured only two cases of Mycobacterium tuberculosis, but this subcutaneous injection of guinea pigs infected with TB specimens of 12%. M. tuberculosis described in the blood than most general bacteria spread type, but a type L Growth difficult. In recent years, many new reports of extrapulmonary tuberculosis, Mycobacterium tuberculosis bacteria detection rate than L-type: such as children 10 cases of tuberculous meningitis cerebrospinal fluid culture, nine cases of cultured L-type, only one case of bacterial type. Difficulty urinating senile prostatic hypertrophy, postoperative pathological acid-fast bacilli accounted for 61.2% of the L-type, no one was typical acid-fast bacilli. Conventional culture-negative chronic prostatitis, nearly 1/3 of the L-type detection of acid-fast bacilli. Infertile men semen checklist to see acid-fast bacilli 7% acid L-type single-see 14% of L-type electron microscope examination showed adsorbed on the sperm head and tail. L Mycobacterium tuberculosis infection in mice, between 73% saw Leydig inflammation L-type acid-fast bacilli.
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Immunity
Immune mechanisms Mycobacterium tuberculosis is an intracellular bacterial infection, the immune T cells mainly based cellular immunity. T cells can not directly and intracellular bacteria, and the infected cells must first reaction, leading to the collapse of the cells, release of Mycobacterium tuberculosis. Although the organism Mycobacterium tuberculosis produce antibodies, but antibody only contacts with the release of bacteria from a supporting role. Mycobacterium tuberculosis invades the respiratory tract, due to the alveoli of 80% to 90% macrophages, 10% lymphocytes (T cells accounted for the majority); primary alveolar macrophages not activated antimicrobial activity is weak, can not prevent swallowed growth of Mycobacterium tuberculosis, anti-tuberculosis can be brought to his office. But presenting antigens, so that the surrounding T lymphocyte sensitization. Sensitized lymphocytes may produce a variety of lymphokines, such as IL-2, IL-6, INF-?, Their interaction with the TNF-α to kill M. tuberculosis lesions. Lymphokines in INF-? Is a major, there are a variety of cells to produce INF-?, Infiltration has for NK,? / ΔT and CD4 +, CD8 + α / βT cells. Some of these cells can directly kill target cells, and some produce lymphokine-activated macrophages that cause respiratory outbreaks phagocytosis strengthened, resulting in the generation of reactive oxygen and reactive nitrogen intermediary intermediary rather kill the germs. Body T cell by antigen receptor (TCR) can be divided into two kinds of different: one from the α chain and β chains, called α / βT cells (including CD4 or CD8 flag), another by a chain and δ? chains, called? / δT cells (CD4 or CD8 mostly no sign). Human and mouse peripheral blood of the former? 90%, which is <10%. Antimycobacterial immunity in these two kinds of T cells play an important role in both. In the early stages of infection α / βT cells have not yet raised the peak of Mycobacterium tuberculosis by? / ΔT cell control. After contact with Mycobacterium tuberculosis? / ΔT cells that proliferate. Healthy people by mycobacterial extract stimulated 7 ~ 10d, peripheral lymphocytes in? / ΔT cells can be increased, its role and α / βT cells also can kill Mycobacterium tuberculosis. In recent years proved that mice infected with Mycobacterium bovis after? / ΔT cells rapidly converge to the area of inflammation, proliferation, mainly V? 9δ2T cell sub_set_s, but people with active TB in this subgroup declined. Recently noticed? / ΔT is a cell-attack primarily the heat shock protein of M. tuberculosis (heat shock protein, HSP). HSP is a highly conserved protein in nature, both from prokaryotes to plants and animals, which have a 50% amino acid sequence homology. In normal organism little content, but infection, fever, and other malignant cells to ambient conditions to produce a large change i.e., also known as the stress proteins HSP (stress protein). HSP has many pathogens, the amino acid sequences have a common component, caused by different bacteria can cause subclinical immune to some extent the same, are considered non-specific immunity. TB infection are immune immune (infection immunity), there are bacteria, also known as immune, have immunity that only when the presence of Mycobacterium tuberculosis or a component when the body. Once the body or components of M. tuberculosis disappeared immune also will not exist.
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Immunity and hypersensitivity
As the body's protective effect of Mycobacterium tuberculosis, but also you can see the generation of delayed-type hypersensitivity, the result both of which are mediated by T cells. You can see from the Koch phenomenon (Koch phenomenon), the M. tuberculosis initial subcutaneous injection of healthy guinea pigs, after 10 ~ 14d local unhealed ulcers, swollen lymph nodes near the bacteria spread to the whole body, the performance characteristics of primary infection. In terms of Mycobacterium tuberculosis to previously infected guinea pigs re-infected, within 1 ~ 2d partial quickly produce ulcers, easy to heal. Nearby lymph nodes enlargement, bacteria seldom spread of infection is characterized by the performance of the primary. When visible light reinfection ulcers, easy to heal, non-proliferation, indicating that the body has a certain immunity. However, re-infection occurs when ulcers faster, indicating that while there is an immune hypersensitivity to participate. In recent years, studies have shown that Mycobacterium tuberculosis induce different substances immunity and hypersensitivity. Tuberculin hypersensitivity is mainly caused by a common protein and waxy D, and immune from Mycobacterium tuberculosis ribosomal RNA (rRNA) cause. Two different antigen components activate different sub_set_s of T cells release a different lymphokine year. Tuberculin test Tuberculin test tuberculin skin test is applied to determine whether the organism Mycobacterium tuberculosis can cause a hypersensitivity test. 1 tuberculin reagents in the past with the old tuberculin (old tuberculin, OT). Department of the Mycobacterium tuberculosis inoculated in glycerol broth, and incubated for 4 to 8 weeks after the heated filter made from concentrate. Diluted 2,000-fold with 5 units per 0.1ml. Currently using purified protein derivative (purified protein derivative, PPD). PPD has two kinds: one Mycobacterium tuberculosis BCG-PPD made PPD-C and BCG made. Each 0.1ml containing 5 units. 2 Test methods and significance of routine tests were taken two kinds of PPD 5 units injected within two forearm skin, swelling and induration 48 ~ 72h after more than 5mm as positive, ≥ 15mm is strongly positive, meaningful clinical diagnosis. If the PPD-C side swelling larger than BCG-PPD side of infection. Conversely, BCG-PPD PPD-C side is larger than the side, which is probably due to BCG vaccination. Negative reaction indicates uninfected Mycobacterium tuberculosis, but should consider the following: ① the initial infection, the need for more than 4 weeks to appear hypersensitivity due Mycobacterium tuberculosis infection; ② elderly; ③ or are suffering from severe tuberculosis patients Other diseases, such as measles immune cells resulting low; ④ get cell immunocompromised, such as AIDS or cancer and other immunosuppressive agents were used. To exclude false negative, and some domestic units plus sterile phytohemagglutinin (PHA) injection, 0.1ml containing 10μg skin test. If the swelling is greater than 24h PHA Picchu by normal cellular immunity, without response or no more than PHA Picchu by immunocompromised. Fourth, microbiological test method Signs and symptoms of TB are often not typical, though can take advantage of X-ray diagnosis, but the diagnosis still depends on the bacteriological examination. _Select_ specimens specimens according infection. Desirable sputum, bronchoalveolar lavage, urine, stool, cerebrospinal fluid, or chest, ascites. Other desirable extrapulmonary infection or blood or tissue cells secrete the corresponding parts. Direct smear smears or _set_s the bacteria smear with acid-fast staining. If the acid-positive bacteria can be found the initial diagnosis. Acid-fast staining is generally used Ziehl-Neelsen method. To enhance staining, the available IK (intensified Kinyoun) staining. The carbolfuchsin stained overnight with 0.5% hydrochloric acid in ethanol bleaching 30s, includes most of the L-type M. tuberculosis can also be colored. To improve the sensitivity of microscopic examination, can also be used auramine staining, fluorescence microscopy was significantly Mycobacterium gold fluorescence. Concentrated _set_ of bacteria strains after checking the first _set_, you can improve the detection rate. Culture and animal experiments must also be _set_ by the bacteria process to remove bacteria. Cerebrospinal fluid and chest, ascites no bacteria directly collecting bacteria can be precipitated by centrifugation. Sputum, bronchial lavage fluid, urine, fecal specimens subject to pollution 4% NaOH (sputum and the base 1:4, urine, bronchial lavage and the base 1:1) treatment 15min, long time easy to make the L-type M. tuberculosis deaths and non-tuberculous mycobacteria. Urine samples before adding 5% tannic acid, 5% acetic acid in each 0.5ml conical cylinder standing, taking sediment treatment. And then the treated material was pelleted by centrifugation. Take sediment smear staining. For further as culture or animal inoculation, should be neutralized with acid and then pelleted by centrifugation. Isolation and culture of the bacteria through the material and _set_ inoculated in solid medium, utensils, plus a rubber stopper at 37 ℃ oral culture, observed once a week. Slow growth of Mycobacterium tuberculosis, which normally takes 2-4 Zhou Changcheng visible off the bacteria. Liquid culture bacteria can be _set_ material dropping in serum-containing medium, you can in 1 to 2 weeks in the bottom of the tube and saw a particle growth. Take sediment smear, can quickly get results, and further for biochemical, and other measuring and differentiating susceptibility of Mycobacterium tuberculosis and non-tuberculous mycobacteria. Domestic scholars have proven Mycobacterium tuberculosis may be present in the L-cells in the blood on the cell surface or adhesion. Such patients often erythrocyte sedimentation rate, immediately after inoculation with hypotonic hemolysis hypertonic saline Mycobacterium tuberculosis L-type medium can improve the culture positive rate. Animal testing material after collecting bacteria inguinal subcutaneous injection in guinea pigs, 3 to 4 weeks after if the local lymph nodes, tuberculin test seroconversion can be dissected. Observed in lung, liver, lymph nodes and other organs or without tuberculosis, and for morphology, cultural and other tests. 6 to 8 weeks if still no disease, should also be dissected checked. Rapid diagnosis is not easy to obtain positive results in general smears bacteria need 5x103 ~ 4/ml, training needs 1x102/ml, the number of samples is less than this number when bacteria and cultures take longer. Currently has polymerase chain reaction (PCR) amplification technology for identification of Mycobacterium tuberculosis DNA, each containing only a few ml of bacteria can get positive, and 1? 2d outcome. Operation should pay attention to the pollution problem of experimental equipment, in order to avoid false positives. Also due to the lack of L-type bacteria and compensatory cell wall thickening, while not commonly used to make cell membranes rupture lysozyme release DNA, resulting in false-negative PCR. Fully with tissue grinders grind the cells rupture, there may be positive. BACTEC conditional units currently used method for containing carbon 14C palmitic acid substrate 7H12 medium, 14C in the measurement produced during the bacterial metabolism of the specimen is projected with acid fast bacilli, 5 ~ 7d can the report. In recent years, domestic and foreign clinical studies have shown that various types of tuberculosis patients in about 40% of the isolated L-type. After treatment of TB bacteria type disappears, L-often persist. There are holes in sputum of patients who have non-exclusive type of bacteria, may be detectable in about 8% of the L-shaped. Therefore, some scholars have suggested that L-type also detected several times as one of the criteria of TB activity, bacterial-type and L-type as sputum were negative to negative. Fifth, the principle of prevention Prevention of nearly 20 years of international organizations to control tuberculosis main methods are: ① detection and treatment of sputum-positive; ② neonatal BCG vaccination. Approximately 80% of the protected force. 1940s survey of part of the city of tuberculosis mortality 200/100 000 or more. After the liberation of improved sanitation, mortality from 1973 to 1977 display has been reduced to 30/100 000. But since 1979, three large-scale random checks nationwide epidemic declined very slowly. From 1979 to 1990 the annual prevalence rate of 2.8% descending, descending sputum-positive rate of 3.0%. Currently mortality 19/100 000, and the other is still two times communicable diseases. Ministry of Health requested in 2000 90% of neonatal BCG vaccination. According to the statistics vaccinated newborns who later than unvaccinated incidence decreased by approximately 80%. BCG is a live vaccine, vaccine directly affects the number of viable cells within the immune effect, so there are already freeze-dried vaccine supply. The new ribosomal RNA (rRNA) vaccine has been cause for concern, but still in the experimental stage. Treatment of rifampin, isoniazid, ethambutol, streptomycin for the first-line drugs. Rifampin and isoniazid may reduce the development of drug resistance. Severe infections can pyrazinamide and rifampin and isoniazid. 1g cheese kitchen or voids containing about 106 to 10 Mycobacterium tuberculosis. 1-6 per 105 may have one kind of bacteria resistant mutation, the need for two kinds of bacteria resistant to 1011, it is appropriate to the two drugs in combination.