Of the many species of Mycobacteria, three are responsible for human tuberculosis. Besides Mr. tuberculosis, the most common, there are cases due to M. bovis or M. africanum.
1865: Villemin shows that human tuberculosis is transmissible by inoculation of rabbits and guinea pigs.
1882: Discovery of Mycobacterium tuberculosis (M. tuberculosis currently named), clotted bovine serum in culture.
1889: Discovery of avian tuberculosis bacillus.
1902 Dorset developing a culture medium egg will be improved by various authors (Lowenstein Jensen, Coletsos, Petragnani).
1902: Discovery of M. bovis, the agent of bovine tuberculosis.
1921: Calmette-Guérin get a vaccine, bacillus Calmette-Guerin (BCG), after 13 years of subculture of a pathogenic strain of M. bovis on potato biliée glycerin.
1944: Waksman discovered streptomycin.
1950: Discovery of the potential role of other pathogenic mycobacteria “nontuberculous” called atypical.
1968: Description of M. africanum.
I – MORPHOLOGY:
A – Optical Microscopy:
D is a bacterium from February to May mcg long and 0.3 microns wide, straight or more or less curved, with rounded ends and motionless. This bacillus is not encapsulated, non-spore forming.
– In pathological products it is in isolated or in small clusters.
– In cultivation can be observed or filamentary coccoid forms.
– A color:
1. This is easily colorable bacilli by conventional dyes.
2. ZiehI-Neelsen staining and auramine are specific to mycobacteria. The latter contain in their wall mycolic acids which are lipid structures responsible for the property “acid-alcoolorésistance” bacteria.
– In the case of the coloring ZiehI Neelsen, the dye used is carbol fuchsin; Mycobacteria appear pink on blue microscopy immersion.
– In the case of auramine staining 0, colorful bacilli have a yellow-green color fluorescence microscopy after excitation at 434 nm.
B – electron microscopy:
The structure of the bacteria is similar to that of other bacteria; among quiescent bacteria found granulations polyphosphates and poly-P-hydroxybutyrate. The DNA of the growing bacteria is associated with mesosomes.
II – CHARACTERISTICS OF CROPPING BACILLI TUBERCULOSIS:
– It is strictly aerobic bacteria sometimes microaerophilic (M. bovis or M. africanum) and then sinking into the culture medium.
Culture is slow (3-4 weeks for M. tuberculosis, 45 to 60 days for M. africanum and M. bovis) the generation time is about 20 hours the culture media. Growth is slower for some strains, particularly those resistant to INH (isonicotinic acid hydrazide).
– These are usually colonies R (eugenics), cauliflower, cream for Mr. tuberculosis or S (dysgoniques) to M. bovis andM. africanum.
The appearance of colonies R is due to the presence of bacteria grouped into strings, and diffract the light and make the opaque colony. In contrast, the S colonies (smooth) have a homogeneous texture, allowing the passage of light;therefore these colonies are translucent.
Optimum growth temperature: 35 to 37 ° C.
Optimum pH 6.8 to 7.0.
Moisture is necessary to culture and the CO2 (5-10%) on agar media.
– Source nitrogen asparagine or glutamic acid.
– Carbon source: glycerol (0.75%) to M. tuberculosis. sodium pyruvate (0.48%) for Mr. bovis.
– Salts (phosphates, potassium, magnesium, iron citrate)
The fatty acids of the culture medium have an inhibitory action on bacterial growth. This action may be lifted by diluting the inoculum in an albumin solution.
A – Solid media with egg coagulated:
They contain 0.025% malachite green for inhibiting the growth of contaminating microbes. For very contaminated samples may be employed with added antibiotics (ac. Nalidixic and penicillin G). In France, the most widely used of these media are mid-Jensen (or Lowenstem-Jensen) and that Coletsos. All are opaque.
1. Löwenstein-Jensen medium is the reference for determining the nature of Eugonique or Dysgonique colonies:
M. tuberculosis forms rough colonies (R) (eugoniques) rough, brittle, cauliflower, opaque, difficult to emulsify, beige-cream and is easily detached from the culture medium.
M. bovis shape of smooth colonies (S) (dysgoniques), small and easy to emulsify.
M. africanum is usually Dysgonique with an acuminate center.
The latter two species grow best on this added pyruvate medium.
2. The medium of Coletsos often gives larger colonies than the Jensen medium. M. bovis grows easily there because this medium contains pyruvate and more glycerol.
It describes three types of Coletsos environments:
– The 4% gelatin for aerobic bacteria and in “good condition”
– One in 20% gelatin to rather microaerophilic bacteria,
– The addition of pulp monkey organs.
Only the first two are currently used.
The advantage of the egg environments is their sensitivity, the characteristic appearance of colonies and their low cost.
Their disadvantage is their variable quality and opacity which does not allow early to observe the appearance of colonies.
B – solid agar media (media from Middiebrook and Cohn: 7:10 ET 7:11)
They are transparent media that should be incubated in 5 to 10% CO2 in plastic bags to retain moisture.
Advantages: transparency can be observed early, microscopy at low magnification, the appearance of mycobacterial colonies and their morphology; This is useful in the case of mycobacterial mixture.
Disadvantages: these environments are contaminated easily and give fewer positive results than with egg circles. In addition their cost is high.
For the growth of M. bovis can also enrich these environments pyruvate.
C – Liquid media:
1. Medium Sauton (mineral salts, asparagine, glycerin):
M. tuberculosis growing in 8 to 10 days in the form of veil.
This medium is used for transplanting BCG strains
2. Mid-Dubos and mid Youmans:
The bacteria settle to the bottom of the tube
3. Middle 7H9 (derived from Dubos medium)
III – IDENTIFICATION CHARACTER:
A – biochemical characters:
1. Production of nicotinic acid (niacin-test):
M. tuberculosis release of nicotinic acid in the culture medium, without the use (99% strain) (tested after 28 days of culture). M. bovis BCG does not release. Mr. africanum can produce more or less early or not at all.
This research is performed between the 28th and the 42nd day of culture.
2. Reduction of nitrate to nitrite (technique Virtanen)
The test is performed on a culture aged 3 to 4 weeks.
M. tuberculosis is nitrate (+),
M. bovis is nitrate (-),
M. africanum is variable nitrate as the techniques used and according biotypes.
All mycobacteria possess catalase, except strains of M. tuberculosis and M. bovis resistant to more than 10 ug / ml of INH.
Fundamental property: this catalase is destroyed by heating at 68 ° C, and at pH 7 for 20 minutes at M. tuberculosis, M. bovis, M. africanum and BCG, and among 3 species of nontuberculous mycobacteria. M.gastri M. haemophilum, M. malmoense (sometimes M. chelonae and M. avium-intracellulare) All other Mycobacteria have a thermostable catalase.
M. tuberculosis hydrolyzes urea, nicotinamide and pyrazinamide.
M. bovis n hydrolysis as urea.
5. 13-glucosidase and lipase:
These enzyme activities exist in M. tuberculosis, but not M. bovis.
B – sensitivity to substances that affect the metabolism of these bacteria:
1. The hydrazide of thiophene-2-carboxylic acid or TCH 2 ug / ml.
All are resistant mycobacteria (less than 1% of survivors), including M. tuberculosis. Only M. bovis and BCG are susceptible. M. africanum is susceptible or resistant.
Note that the INH resistance determines cross-resistance to TCH M. bovis.
2. Thioacetazone or thiosemicarbazone Tb] 10 fjg / ml M. tuberculosis and M. bovis is sensitive to 10 mcg / ml Tbi while most non-tuberculous mycobacteria (except M. kansasii, M. and M. scrofulaceum gastric) are resistant.
M. africanum may be susceptible or resistant.
3. Mr. pyrazinamide tuberculosis and M. africanum are sensitive and M. resistant bovis.
IV – PHYSICAL AND CHEMICAL SENSITIVITY TO AGENTS:
A – Physical agents:
– Temperature: These seeds are sensitive to heat and this property is used in the pasteurization of milk to destroy Mr.bovis (63 ° C for 30 min).
By resisting against these bacteria at 4 ° C.
– Light: tubercle bacilli are sensitive to ultraviolet rays.
– Desiccation They are also resistant to desiccation and remain virulent in the dried droplet Pfltigge.
Lyophilization conservation strains.
B – Chemicals:
Acids and bases destroy mycobacteria, but less quickly than ordinary germs. This property is utilized to decontaminate some samples (sputum, urine) while maintaining the viability of mycobacteria.
2. isopropyl or ethyl alcohol:
These alcohols destroy the TB germs in minutes; they are used on the skin.
3. soap-phenol mixtures using o-phenylphenol:
Mycobacteria are killed within 10 to 30 min. The residual activity is 2 to 3 days.
These products can be used on the skin.
4. The sodium hypochlorite:
It is effectively diluted to 1 / 200th with 10 to 30 minutes of contact, but it has no residual action.
5. The formaldehyde 3-8% alkaline glutaraldehyde at 2% and 5% phenol are also active.
6. The quaternary ammonium compounds have no effect on mycobacteria.
This property is exploited in some decontamination protocols biological samples.
V – CHEMICAL CONSTITUTION:
Mycobacteria are the richest bacteria extractable fat: 20 to 45% of the dry weight of the bacteria. Their basic structure is a peptidoglycan covalently bound to a mycolate arabinogalactan.
Its composition is analogous to the peptidoglycan of other bacteria.
This polysaccharide is characteristic of bacteria of the genera Mycobacterium, Corynebacterium, Nocardia.
This is a hapten.
3. mycolic acids:
These are branched fatty acids and hydroxylated formed from 60 to 90 carbon atoms.
These mycolic acids are responsible for the acid-resistance of mycobacteria.
4. mycolate arabinogalactan:
Lipopolysaccharide bacilli Gram (-) is replaced by the mycolate arabinogalactan.
Are glycolipids and peptidoglycolipides having in common a terminal saccharide containing rhamnose 0-methylated.
They are associated with the wall of mycobacteria and are specific for certain strains:
– Type A: potentially virulent strains of nontuberculous mycobacteria (M. kansasii) and Mr attenuated virulence oftuberculosis.
– Type B strains M. bovis.
– Type C avian strains or saprophytes.
6. The “cord-factor” (consisting of mycolic acid, trehalose, trehalose dimycolate):
He is responsible for the formation of “serpentine cords” M. tuberculosis in liquid medium Youmans: these clusters bacilli grouped into strings at Ziehi staining.
The presence of these formations is correlated with the virulence of tubercle bacilli.
The cord-factor does not cause TB immunity after injection.
They are extractable wall elements by alcohol-ether. According to their physicochemical properties are distinguished four types: A, B, C and D. The virulent strains possess 6-8% waxes D; these can replace mycobacteria in Freund’s complete adjuvant which is used as an adjuvant factor of humoral immunity in the animal.
Topologically, the bacterial cell wall is formed from the inside to the outside of the cytoplasmic membrane, peptidoglycan, polymer arabinogalactan and mycolic acids involved in trehalose dimycolate.
They are the support tuberculin activity and is extracted from the bacterial body or culture filtrate. Three types of preparation contain:
– Old tuberculin Koch which is a concentrated culture ultrafiltrate consisting of proteins, polysaccharides and nucleic acids,
– Tuberculin IP-48, consisting of carbohydrates and protein,
– PPD = “Purified Protein Derivative” Seibert formed only of protein and which serves international reference standard (RT = 23).
These proteins injected intradermally used to check whether a subject has been in contact with Mr. tuberculosis orM. bovis:
– If the subject was never tuberculisé, we do not get a local reaction (or skin infiltration, redness or papule)
– If the subject was tuberculisé, these proteins cause a local delayed hypersensitivity reaction (HSR). This reaction is identical with the different Mycobacteria tuberculosis. There are cross-react with similar proteins extracted from other mycobacteria and called sensitins.
VI – GENETICS M. TUBERCULOSIS:
A – Mutants resistant to antibiotics:
1. Mechanism of resistance:
The resistance is due to chromosomal mutations; no transfer of genetic material leading to a resistance could be highlighted.
Resistance mutation rate:
Isoniazid, or isonicotinic acid hydrazide (INH): 5.10-6
2. Characters of INH-resistant mutants:
a / Resistance to 0.1 or 0.2 ml INHI jig is related to:
– Resistance to high concentrations of ethionamide,
– The persistence of catalase activity,
– The persistence of virulence for guinea pigs.
b / f resistance have alo g INHIml X is related to:
– The persistent sensitivity to ethionamide,
– The loss or significant decrease in catalase activity,
– A large decrease in virulence in the guinea pig.
3. Characters of ethionamide-resistant mutants:
a / Some resistant Tb]
b / Others remain sensitive Tb, but resistant to low levels of INH (0.1-0.2 ug / ml).
B – Genetic Variations Mr. tuberculosis:
– Hungary: green colonies on Löwenstein-Jensen sensitive to TCH.
– Asia: dysgoniques colonies.
– South India, Madagascar: 30% of strains with low catalase activity and virulence for guinea pigs decreased and are very resistant to INH and Tbi.
– BCG varying Mr. bovis.
C – Existence many mycobacteriophages:
12 bacteriophages distinguish phage 5: 3 main types (A, B and C) and two subtypes (Ax and A2).
There are geographical differences in phage.
VII – PATHOGENICITY HUMANS:
The man is very susceptible to TB infection, but only 3-5% of those infected will develop pulmonary tuberculosis; this percentage increases in subjects stressed or living in a confined environment.
Tuberculosis is a disease still widespread; 7,000,000 subjects are contagious in the world, of which 3/4 in developing countries.
Each year 3.5 million new infectious cases are identified and the number of people infected each year is estimated 5 to 8 million. About 2 to 3 million people die from TB each year.
The annual incidence of new cases of tuberculosis – disease (incidence =) varies by country:
– 300 to 500 cases per 105 inhabitants in selected countries in Asia, and Oceania,
– 250 cases per 105 inhabitants in Africa,
– 25 cases per 105 inhabitants in France (1987)
– Less than 20 cases per 105 inhabitants in Australia, Canada, Denmark, USA, Netherlands.
She declined steadily throughout.
In France in 1910 to 288 105 inhabitants; 1950: 58 105 inhabitants in 1974: 6 105 inhabitants
Annual infection rate: Percentage of population positivant its cutaneous tuberculin each year: 1975: 0.1% in France, 0.01% in the Netherlands, about 2% in developing countries.
In industrialized countries the rate decreased 10 to 11% every year since 1950, but it is common in developing countries. The fall in incidence observed in developed countries mainly due to the improvement of living conditions, milk pasteurization, early diagnosis, treatment of patients and chemotherapy of contacts.
Currently in France, 99% of human tuberculosis due to M. tuberculosis and 1% Mr. bovis. As for Mr. africanum, it can be met in West Africa mainly.
It develops in succession: an inoculation chancre, lymphadenopathy and possibly hematogenous spread of bacilli in all organs.
1. The canker of inoculation:
Inhaled bacilli (the most common) preferentially arrive in the middle lobe and the right lower lobe and sink to the level of the cell, just under the pleura. This produces a local inflammatory reaction nonspecific: alveolar macrophages engulf the tubercle bacilli; they continue to multiply in the cell and destroy; they are freed, taken over by other macrophages, and the cycle repeats. It therefore makes inoculation chancre alveolitis.
Bacilli spread of this primary focus to loco-regional lymph nodes, that is to say the tracheobronchial lymph nodes and multiply there, creating nodes. The infection spreads to other nodes by lymphogenic track.
At the same time develops the cellular immune response in an attempt to limit the spread of bloodborne germ.
3. Dissemination blood-bacilli throughout the body:
By efferent lymph bacteria can win the general circulation and reach all organs.
B – Evolution of primary tuberculosis:
This is the most common case (95% of cases), because during the different phases of primary tuberculosis develops the subject hypersensitivity to tuberculin (4-12 weeks). This hypersensitivity is associated with stimulation of cellular immunity by increasing efficiency vis-à-vis the tubercle bacilli macrophages.Then observed histologically the formation of tubercle follicle around the lesion alveolitis: macrophages differentiate into epithelioid cells and multinucleated giant cells known as “Langhans’ which have follicles around the lesion that caséifie. The grouping of several follicles makes macroscopically visible tubercles, which gave their name to the disease.
The bacilli in the organs are, or sometimes destroyed and healing is ultimately more often quiescent and help maintain a population-specific T-memory cells to prevent re-infection with this organism.
However, these germs can reactivate quiescent (stress, cortocoïdes, irradiation, immunosuppression). Specific immunity is very imperfect.
2. TB disease:
It is sometimes said immediately after the primary infection, or several years later. In our regions it is most often due to wake quiescent bacilli: the tuberculosis endogenous reactivation. Sometimes the disease is triggered by superinfection with a new strain of TB: the tuberculosis infection.
During active disease, organ can be reached (kidney or joint) without pulmonary involvement. In infants are more readily observed tuberculous meningitis and miliary; adolescence serous will be reached more frequently while pulmonary locations will be seen preferably in adults.
In the lung, where the casein is poorly oxygenated (casein or casein softened still solid non vented), the number of bacilli decreases; when the casein is in large part removed (caves bronchus open drain), oxygenation is satisfactory and the high number of bacilli. The majority of lesions in the pulmonary tuberculosis appears to be due to the toxic action of the macrophages activated by T lymphocytes These activated macrophages release also many cytokines (interleukin-1, TNF) which may have an immunoregulatory role locking the T cell response (hence chronic infection), impaired lymphocyte recirculation (hence low dermal delayed hypersensitivity reaction and decline of specific immunity).
C – Transmission of tuberculosis:
M. tuberculosis is a strict parasite of the human species, man is both the tank and the bacillus transmission agent.Patients with pulmonary cave are the main disseminators of the bacillus through droplets released during speech, coughing or étemuement.
Infections other than lung, have a much lower epidemiological role.
Animals infected with human contact (dog, cat, monkey) little involved in the spread of the disease.
Human infection with M. bovis is predominantly made by the ingestion of contaminated dairy products. Human lung attacks on Mr. bovis can be a source of contamination for other subjects.
VIII – EXPERIMENTAL PATHOGENICITY:
A – Animal choices: guinea pig:
It is sensitive to M. tuberculosis and M. bovis and all routes of inoculation are effective; moreover it is not susceptible to germs can contaminate crops (Bacillus). The products are decontaminated with sodium hydroxide at 0.25% (final concentration), then neutralized injected. In practice, a subcutaneous injection is carried out in the inner thigh in a guinea pig does not react to tuberculin
– 10 to 15 days after injection appears that a nodule forms a chancre ulcer that persists until the death of the animal,
– And appears satellite lymphadenopathy which extends to all nodes,
– Finally, the death of the animal occurs in one to three months.
At autopsy, whitish nodules were observed in the spleen, the liver and the lungs; these organs contain tubercle bacilli.
However Mr. tuberculosis and M. bovis resistant to 10 ug / ml of INH are highly attenuated for virulence in guinea pigs. BCG has no pathogenicity for guinea pigs; However, at very high doses appear lesions that heal quickly.
This inoculation was not performed because the current methods in vitro are satisfactory and guinea pigs are insensitive to other potentially pathogenic mycobacteria in humans.
B – The Rabbit:
It is sensitive to 10 pg of M. tuberculosis and M. africanum injected intravenously: it creates lung injury often insignificant and regressive.
By cons, 10 ug of M. bovis caused by the intravenous route, the death of the animal with generalized tuberculosis.
C – The mouse:
It is susceptible to M. tuberculosis and M. bovis by intravenous route.
D – The phenomenon of Koch:
II is a phenomenon demonstrated only in guinea pigs. The injection of subcutaneous tubercle bacilli by a product of late blight apparititon inoculation (15th day). A second injection of tubercle bacilli is then performed in the same way in another site.
1st case: two injections separated by 40 days or more
– There is a suppurative canker first injection site; it will remain until the death of the animal, and the lesion evolves slowly and gradually with dissemination of bacilli throughout the body of the guinea pig.
– The second point of injection ecchymotic is within 24 to 48 hours, necrosis and forms a superficial ulcer heals without mycobacteria in 10 to 15 days. It is a strong and early development. This second inoculation does not develop as the first one.
Case 2: the two injections are separated by less than 25 days
In this case, the animal does not yet of sensitivity to tuberculin and the second inoculation is changing as the first.
Koch phenomenon thus shows the presence of two reactions in the body:
– The sensitization phenomenon (bruising and eschar)
– The phenomenon of resistance (blocking the multiplication of bacilli and stopping their dispersion).
IX – BACTERIOLOGICAL DIAGNOSIS OF TUBERCULOSIS – SICK:
A – Direct diagnosis:
It is the demonstration of bacilli by microscopic examination and by culture.
1. Sampling and treatment:
a / Withdrawals:
– Coughing morning (5-10 ml) obtained during coughing or after spray of salt water to 10%.
– Casings stomach before breakfast (they correspond to swallow sputum overnight). If these casings must wait more than 4 hours before being processed, it must be added 100 mg of NaHC03.
– Urine collected in the middle of the jet (morning urine and not 24 hours).
– Swabs can be a source of false negative results due to the small amount of material collected and the difficulty of detaching mycobacteria swab (role of lipids of the bacterial envelope).
– Blood drawn either in a tube containing saponin (DuPont Isolator System) to release the mycobacterial phagocytes, or in a tube with anticoagulant when using the radiometric culture system.
– Fabrics, pleural fluids, peritoneal, blood, pus, joint fluid.
All samples must be kept at 4 ° C, especially if a certain time must elapse between sampling and processing by the laboratory.
b / Treatment:
– Sterile Products (LCR, punctures, biopsies, blood hemolysis with saponin):
Focus bacilli by centrifugation if the products are liquids; for solid products, grind in a mortar with sand and centrifuge after settling for a base to be used for direct examination and culture.
Concentrate urine by centrifugation; make a blade with a fraction of the base; the remaining pellet was decontaminated as either sputum or by H2SO4 (2% final) for 15 min and neutralized.
We must standardize, that is to say make them smooth and eliminate mundane germs. Describes many techniques:
– The Petroff technique (soda 2% final) at 37 ° C for one hour with stirring,
– The Tacquet and Tison technique (soda 0.5-0.7% and 1.5-2% SDS) for 40 minutes stirring at room temperature,
– The technical benzaikonium chloride (quaternary ammonium) phosphate trisodium for 30 minutes stirring,
– The technical to acetylcysteine-soda for 15 minutes stirring,
– Technology cetyl pyridinium (quaternary ammonium)
– Technical oxalic acid 2.5% contaminated sputum by Pseudomonas.
After this treatment, the pellets obtained after neutralization and centrifugation, are deposited on a slide for direct examination and seeded.
2. Direct examination:
A fraction of the base is spread on a slide and stained.
– Rapid Screening Method for fluorescence microscopy: the auramine staining 0 (objective 40, fluorescent yellow-green bars). The same blades can be stained with Ziehl for confirmation of a positive result.
– Coloring ZiehI-Neelsen carbol fuchsin in classical microscopy immersion. A blade should be observed 20 minutes (target 100, rosé sticks) before concluding the negativity of the review.
The abundance of acid-fast bacilli observed to be expressed in the form of a semi-quantitative result.
a / solid media:
One seeded Löwenstein-Jensen tubes and / or Coletsos with pellets.
Products for sowing can be suspended in 1-2 ml of albumin 2% o pH 6.8 to promote the cultures.
Usually seeded, several tubes to the egg circles, out.
Some authors recommend further seeding agar medium allowing early diagnosis and medium egg containing antibiotics (nalidixic acid, penicillin) for contaminated samples. The whole medium surface must be sown; must be plugged with cotton or do not screw tightly the cap to dry the inoculum for 24 to 72 hours at 37 ° C. Finally, we must recap or tightly screw the tubes to avoid desiccation of the medium. These cultures were kept three months at 37 ° C.The agar media are incubated in the presence of 5-10% CO 2 in plastic bags.
The original surface samples will be incubated four months at 30 ° C and 37 ° C for the monitoring of certain non-tuberculous mycobacteria (M. marinum, M. ulcerans, M. haemophilum).
The first reading was made in the first week to remove contaminated tubes (up to 5%, the infection rate is normal and acceptable) to identify and rapidly growing mycobacteria. Tubercle bacilli grow in three weeks or more, so the crops will be kept for about 3 months and regularly checked.
b / Liquid media:
Mycobacterial culture can be carried out directly in a liquid medium. The sterile sampling and decontaminated by treating those are seeded in sealed vials containing a liquid medium Middiebrook added with a combination of antibiotics and antifungals (polymyxin B, amphotericin B, nalidixic acid, trimethoprim and agiocilline).
In the radiometric method (type Bactec), a liquid medium is used 7 H12
added palmitate 14C; for blood cultures, this medium is still
supplanted. Was measured 2-3 times per week increased 14CO2 in the atmosphere of culture. The growth of tuberculosis mycobacteria is detectable on average by the end of the first week of culture. The flasks are monitored for 6 weeks.
Another non-radiometric method uses a two-phase medium. Covered blades agar media (chocolate agar medium and 7:11 7H11 medium supplemented with P-nitro and acetylamino hydroxypropiophenone p) are immersed in a regular turnaround 7H9 liquid medium. The bacterial growth is observed on average in 2 weeks.
4. Inoculation in guinea pig:
The guinea pig inoculation is rarely used because the current culture media are more efficient.
It is sometimes useful to purify mycobacterial cultures contaminated with Bacillus or in the case of highly contaminated products (then add antibiotics to products homogenized with sodium hydroxide).
It is mainly used to confirm the pathogenicity of a mycobacterium.
5. Identification of mycobacteria in culture:
We must proceed in order:
a / Ziehl:
Check that the colonies, pigmented or not, fast-growing or not, are acid-fast bacilli.
b / Growth rate at 37 ° C and growth temperature:
M. tuberculosis, M. bovis, M. africanum, BCG grow slowly (3 weeks or more). The assessment of the growth rate is made on a subculture colonies isolated.
c / Pigmentation and appearance of the colonies:
Some mycobacteria are pigmented yellow-orange, or in the dark (scotochromogenic) or only after exposure to light (photochromogens). Tuberculous mycobacteria are cream.
The rough or smooth colonies can direct identification.
d / biochemical reactions and resistance to antibacterial agents:
a. positive diagnosis:
Eugoniques colonies (R) usually
Nitrate (+) (Virtanen technique)
Catalase 68 ° C (-)
Dysgoniques colonies (S) to very slow growth (6 weeks)
Catalase 68 ° C (-)
Eugoniques colonies like those of M. tuberculosis
Biochemically it comes to Mr. bovis resistant D-cycloserine and sensitive to other anti-tuberculosis antibiotics.
Similar growth rate to that of M. tuberculosis
Dysgoniques colonies with acuminate center Löwenstein-Jensen
Niacin (+/-) or delayed positivity (42nd day of culture)
Nitrate (- / +)
TCH and Tbi: susceptible or resistant catalase at 68 ° C (-)
It is often a discrepancy between the number of bacilli seen on direct examination and the low number of colonies observed to culture.
M. africanum has intermediate characters between M. tuberculosis and M. bovis. It grows slowly and gives dysgoniques colonies on Löwenstein-Jensen. We distinguish three types according to their biochemical reactions (reduction of nitrate according Tacquet) and resistance to antibacterial agents TCH and Tbi. Kinds Rwanda, Dakar and Yaounde Mr. africanum is observed in 30-40% of tuberculosis of West and Central Africa.
b. differential diagnosis:
– Mycobacteria Niacin (+) M. tuberculosis, M. simiae (scotochromogène), sometimes Mr. marinum(photochromogen), sometimes Mr. chelonae (fast growth).
– In the presence of slow-growing mycobacterium, non-pigmented, niacin (-), nitrate (-) and catalase 68 ° C (-), we must mention: Mr. bovis BCG, M. gastric Mr. malmoense and sometimes Mr. avium-intracellulare whose activity catalase at 68 ° C is often low. Sensitivity TCH is one of the characters to differentiate M. bovis, BCG should be discarded by the research of the sensitivity to D-cycloserine and / or by the inoculation of guinea pigs.
c / Presumptive Identification direct liquid culture:
In Bactec system, bacteria grown in liquid medium were transferred to another flask added NAP compound (p-nitro and acétylmino, hydroxypropiophenone). The growth of bacteria of tuberculosis is inhibited by this compound, while that of the other mycobacteria continues to perform. This growth is compared to that which occurs in another bottle free of NAP.
The growth rate is further studied in the liquid medium (slow enough mycobacteria tuberculosis) and the morphological properties and tintoriales mycobacteria. This differentiation between tuberculous and non-tuberculous bacteria is of clinical and epidemiological importance because of its earliness. However, BCG is not differentiated from M. tuberculosis and additional methods should refine the identification.
d / identification using specific probes:
Probes specific nucleic acids of group tuberculosis from M. avium-intracellulare, of M. kausasii and M. gordonaeallow the identification of some species have grown in solid or liquid media. They are useful for differentiating tuberculous mycobacteria nontuberculous and the results are very satisfactory. They even allow to detect tuberculous mycobacteria in samples rich in bacilli. Their disadvantage is still their radiolabeling.
6. Study of the anti-tuberculosis antibiotics:
a / Conventional method:
The clinical efficacy of an antibiotic depends on the proportion of resistant mutants naturally present in the strain. The study in vitro sensitivity of a strain to tuberculosis substances is carried out by the ” proportion method ” carried out in solid medium and to detect the rate of each antibiotic resistant mutants within the strain to be studied.
In current practice using the proportion method of Canetti, Rist and Grosset: a susceptibility box consists of three identical sets of circles, each formed of two Lowenstein-Jensen media tubes as controls and several tubes of the same medium added to a concentration of antibiotics (INH, ethambutol …). This concentration is called critical concentration.
Performing a suspension of the test strain in distilled water at a concentration of 1 mg / ml by comparison with a standard, and it performs three dilutions: 10-1, 10-3 and 10-5. Each dilution was inoculated a set of tubes (0.2 ml / tube).
Reading consists in measuring the percentage of resistant bacteria to each antibiotic tested. This percentage is compared to a number called critical proportions which differs according to antibiotics. If the percentage of survivors is greater than the critical proportions, the mycobacterium is considered resistant; if that percentage is inferior, it is regarded as sensitive. Playback is at day 28; for M. bovis it often takes 40 to 50 days before you can read the antibiogram.
Critical concentrations of antibiotics are totally different from the MIC of the antibiotic; critical proportion has nothing in common with the spontaneous mutation rate of resistance to this antibiotic. Critical proportions and critical concentration is typical of TB in a given culture medium. Both numbers were determined on bacteriological bases, pharmacological and therapeutic. The critical concentration depends on whether susceptibility testing is performed on medium egg or agar because the presence of egg proteins leads to a lower bioavailability of certain antibiotics;further heating to 85 ° C for 50 minutes needed for clotting of the egg environment can reduce the activity of certain antibiotics.
A dilution 10-5 is an average of 25 colonies per sample tube. The 10-3 and 10-1 dilutions of the cultures are confluent in the control tubes and the number of bacteria by control tube is estimated at 25 x 102 and 25 x 104 respectively.
The percentage of antibiotic-resistant mutants is calculated as follows:
Rifampicin: 50 / (25 x 104) = 2 x 104 = 0.02%: strain sensitive to rifampicin.
Streptomycin: 110 / (25 x 10²) = 4 x 10-2 = 4%: strain resistant to streptomycin.
This method allows for an indirect susceptibility from the culture of a mycobacterium or direct susceptibility testing from a pathological previously decontaminated, provided that it is sufficiently rich in bacilli (at least 1 to 10 10 fields immersion).
The study of the sensitivity of mycobacteria to pyrazinamide is to be performed at acidic pH (5.5). It is necessary that the control tubes show growth at this pH, which is sometimes difficult to obtain. This is why the control tubes used (without antibiotic) are different from those used to test other antituberculosis antibiotic.
b / radiometric method in liquid medium:
Susceptibility testing of Mycobacterium tuberculosis of the group can be obtained more quickly using the Bactec system (7:12 medium supplemented with a 14C-labeled substrate). The suspension is ensemensée mycobacteria in a control flask containing no antibiotic and in various vials each containing an antibiotic (INH, streptomycin, rifampicin, éthamhitol, pyrajinamide). A seed is considered sensitive to the antibiotic tested whether its growth, followed by the production of 14C02, is inhibited by more than 99% in the presence of the antibiotic as compared to that observed in the absence of antibiotic. The results of susceptibility testing are usually obtained within a week instead of the 3 weeks of the conventional method.
This technique also allows rapid MIC determination of a given TB, studying at least 3 different concentrations of this product.
Radiometric technique has the advantage of speed. It does not however yet to test the sensitivity of the so-called atypical mycobacteria.
7. Rapid and sensitive detection of mycobacteria directly in the pathological products: laPCR
A method of in vitro DNA amplification (PCR polymerase chain reaction) should soon be marketed (1992). It will allow simultaneous detection and identification of mycobacteria directly in pathological products within hours, without going through the stage of culture. Preliminary results compared to culture methods are satisfactory. The advantage of this technique is its speed; its Downside is not to allow the susceptibility testing since no strain is isolated by PCR.
B – Serological diagnosis:
It never entered into practice and shows no diagnostic value today. Indeed there may be antibodies in infected individuals, but it is also in subjects vaccinated with BCG Also it is not yet demonstrated that they are a good marker of infection or no infection or efficacy of antibiotic treatment.
X – TREATMENT:
A – Curative:
Tuberculous cavern comprises about 108 bacilli, therefore spontaneously contains 100 1 000 germs resistant to an antibiotic. So it would take at least two antibiotics to achieve effective treatment. In practice we combines three to four as 5 to 10% of the infecting strains are resistant outset, before any treatment, at least one antibiotic. This is theprimary resistance.
Currently the primary resistance as follows:
– Streptomycin: 6% of the strains,
– INH: 3%
– Rifampicin: 0.2%
– Ethambutol 0.1%.
The primary resistance is stable statistically.
After 2 months of treatment, if crops do every month is negative, we give only two antibiotics because it then has the antibiogram and because the residual bacterial population is about 102 bacteria only. If cultures remain positive after a few months of treatment, it will redo susceptibility testing for secondary resistance will have appeared (poorly monitored treatment).
This secondary resistance tends to decrease.
The line drugs used are:
INH, rifampicin, ethambutol, streptomycin, pyrazinamide (it is idle to M. bovis)
In case of multiple resistance using second-line drugs: NO (para-amino salicylic acid), kanamycin, D-cycloserine, ethionamide, prothionamide, Viomycin, capreomycin.
The duration of treatment is currently of the order of 6 to 9 months in the absence of complications.
Note that the fluoroquinolones (ofloxacin) are active on tuberculosis mycobacteria.
B – Prophylactic treatment:
1. Radiation screening:
Students, hiring (medicine), laboratory workers, doctors …
INH for subjects contact. One antibiotic simply because these subjects are very few bacillus. It is necessary to check if the strain isolated from the patient is sensitive to INH.
3. Control of bovine tuberculosis:
Cases of human tuberculosis M. bovis decrease with the control of bovine tuberculosis and milk pasteurization.
4. BCG vaccination:
II is a strain of M. bovis virulent originally. After 230 passages on potato biliée glycerin, this strain lost its pathogenicity for all animals and man.
The vaccine is prepared from sailing obtained Sauton medium; it is to be crushed and placed in suspension in a liquid medium (stable for 3 to 4 weeks) or lyophilized (better conservation).
– Method of administration
– Buccal (inefficient)
– Intra-dermal injection: very effective technique, although quantifiable but accompanied by 5% large local reaction
– Scarification: very effective technique, few accidents, but imprecise.
Vaccination is performed in subjects with a negative delayed hypersensitivity reaction to tuberculin.
Children are often vaccinated in the first quarter of life, but vaccination is compulsory in France at the age of 6 years.
As this is a live strain, patients with immune deficiency, and pregnant women should not be vaccinated. Other indications against eczema in infants, prematurity, corticosteroids, scalable acute illness.
BCG, once injected, produced in three weeks canker inoculation with satellite lymphadenopathy. Immunity develops at the same time or a little later than the delayed hypersensitivity. The efficiency of vaccination is checked by skin test two months after inoculation.
The immunity conferred by vaccination is imperfect; it is not final either, and reminders may be needed.
The incidents are rare (less than 1% <?)
– Adenitis fistulized spontaneously yields in a few months or ulceration of the vaccine nodule,
– Subcutaneous abscess in case of subcutaneous injection,
– Post-vaccination erythematosus, musculoskeletal disorders (infants) and fatal BCGitis due to immune deficiency ignored during vaccination.
– Effectiveness of BCG:
Since 1921, several hundred million vaccinations were carried out, but the degree of protection afforded by BCG remains the subject of discussion.
The eight major trials have yielded conflicting results: for tests in temperate zones, protection is about 80%; for tests in tropical areas, protection appears to be low or zero.
But in the tropics latent infection by nontuberculous mycobacteria is widespread, resulting in a low degree tuberculin hypersensitivity and therefore some immunity against tuberculosis, sufficient to mask the specific effect of BCG, but not enough to really protect subjects against tuberculosis.
Currently it seems that BCG exerts a protective effect by blocking the hematogenous spread of bacteria, limiting the infection to subclinical proportions. BCG does not prevent primary infection and has no therapeutic role vis-à-vis an incorporated infection; it mostly reduces the probability of occurrence of miliary and tuberculous meningitis, especially in young children. That is why, internationally, we recommend BCG vaccination of all children living in areas where there is a high rate of spontaneous conversion of hypersensitivity reactions to tuberculin.
Finally the use of BCG in certain neoplastic diseases and in the treatment of lepromatous leprosy as nonspecific stimulation means of cellular immunity.
C – laboratory Precautionary Measures:
People working in medical bacteriology laboratories are among the most exposed to the risk of tuberculosis subjects. This is why certain precautions are necessary: in particular, it is necessary to minimize the spread of mycobacteria in air and avoid inhalation of tubercle bacilli.
Staff working with mycobacteria must undergo a medical examination once a year. The delayed hypersensitivity to tuberculin must be checked each year among negative subjects; in others, a chest radiograph should be performed each year.
Platinum handles can be dangerous to use because the liquid film they carry may shatter and disperse germs all around; using Pasteur pipettes is better with a pipetting device adapted Pipetting by mouth should be outlawed.;Hand washing should be careful.
The laboratories will be equipped with hoods (0.4 m / s) protecting the operator with certainty, and under which will be carried out the following: transfer of samples from one tube to another, agitation virulent solutions preparation spreads on blade, planting media. Cotton wool soaked in disinfectant must be available under the hood to clean the edges of the tubes in which were transferred from contaminated solutions. The hood work plan should always be disinfected before and after use.
Centrifuges and mixers must be located in specially ventilated.