Laboratory Diagnostics of the tuberculosis and leprosy
Etiopathogenesis of tuberculosis
Tuberculosis is an infectious disease and its pathogene is a Mycobacterium tuberculosis (MBT) from the genus Mycobacterium of Actinomycetacae family.
Morphology of Mycobacterium tuberculosis
MBT looks like a bacillus from 0.8 to 5 mkm long, from 0.2 to 0.3 mkm thick. MBT can exist in various forms: typical rods, chips, L-forms and filtrating forms.
Groups of mycobacteria
3 groups of mycobacteria are distinguished: true (pathogenic for a human being), atypical and acidic-stable saprophytes. There are three types (species) of pathogenic MBT: human (M. tuberculosis), bovine (M. bovis) and African (M. africanum).
Cord-factor of MBT
Lipoid cell wall of mycobacteria forms its virulence and capacity for education in culture accumulations of bacteria as scythes (cord-factor).
Cord-factor, as be set later, related to the unusual biological matter of trehalose 6, 6-dimycolate, which possesses high virulence.
L-forms of MBT
One of important types of changeability of MBT is forming of L-forms. L-forms are characterized by reduced urovmute metabolism, the loosened virulence. Remaining viability, they can for a long time be in an organism and induce antituberculosis immunity.
Resistance of Mycobacterium tuberculosis in the environment
All of them are very stable in the environment. Particularly, they are preserved in the soil for 1-2 years, in basins - up to 5 months, in the road dust - up to 10 days, in premises at the dissipated sunlight - up to a month and a half, in excrements and on pasture-grounds up to a year, in butter, cheese kept in a fridge - 8-10 months, on books' pages - 3 months. At the temperature of 20° C MBT preserve their vital activity for 7 years. Boiling liquid sputum kills MBT within 5 minutes. Under the action of sun rays MBT die in an hour and a half, and that of ultraviolet radiation - in 2-3 minutes.
Sourse of infection the Tuberculosis
Sick people and animals, secreting MBT, are the source of human tuberculosis infestation. A pathogene, depending on the affected organ, is secreted into the environment with sputum, excrements, urine, milk, sperm etc. Infectation occurs most often by aero-genic (90 %), contact (5-6 %), rarely alimentary (1-2 %) and extremely rarely by intrauterine way.
Matherial for microbiological research of Tuberculosis
The materials for revealing MBT are sputum, bronchial lavage waters, faeces, urine, fistula pus (matter), pleural cavity exudate, spinal fluid, punctates and bioptates of various organs and tissues.
Principle microscopic methods for diagnosis of Tuberculosis
Bacterioscopy is one of the main methods of revealing MBT; it includes ordinary bacterioscopy, flotation and luminescent microscopy. An ordinary bacterioscopy is accessible to everybody, it is simple and quick to do. In smears, coloured according to Zeihl-Nilsen, MBT are revealed when not less than 50000 microbic bodies are present in 1 ml of pathologic material. Under the microscope MBT look like bacilli of the red colour on the blue background.
Flotation method (enrichment or concentration of MBT in a small volume, caused by droplets of benzine, benzole, xylol or toluene on the surface of a retort ring) is applied in cases when there is a small number of MBT in the pathologic material and at negative results of ordinary bacterioscopy. Flotation method provides for 10-15 % more often revealing MBT in comparison to direct bacterioscopy.
Luminescent microscopy is based on the ability of MBT, coloured (painted) with fluorochroms, to illuminate under the influence of ultraviolet rays and performing microscopy at small magnification, increasing by 15-30 % the sensitivity of the method in comparison to direct bacterioscopy and by 10 % in comparison to the flotation method.
Principle of bacterilogical method for diagnostics of Tuberculosis
Bacteriological method consists in the following: sputum or another material, after preliminary special treatment, is inoculated on nutritive media (hard, blood, semisynthetic). More often hard egg Löwenstein-Jensen medium is used. 20-100 microbial bodies in 1 ml of sputum is enough for revealing MBT culture. The first colonies appear on the 18-30-th day of cultivation. The negative result is given only in 3 months from inoculaion. This method of revealing MBT allows to define their vitality, virulence, group (differentiate from acid resistant saprophytes and atypical MBT) and species origin, as well as their resistance to antimycobacterial preparations. In addition to this, according to the data of bacteriological examination quantitative essessment of bacterial secretion is made: miserly-up to 10 colonies on a nutrient medium, moderate - from 10 to 50 and massive - more than 50 colonies.
Principle of biological method for diagnostics of Tuberculosis
It is infection with sputum or another pathologic material of guinea pigs, which are highly sensitive to MBT. A biological testing is the most sensitive method of revealing MBT, as far as in laboratory animals tuberculosis develops after the introduction of the material in which there may be less than 5 microbial bodies in 1 ml. However, it should be noted that MBT are stable to chemical preparations, particularly to isoniazidum, avirulent for guinea pigs. That is why various methods of microbiological examination should be used for revealing MBT in pathological material. Generally, before starting to treat a patient, he should undergo complex bacteriological examination. It is three times direct bacterioscopy of sputum or, when it is absent, three times examination of the material after provoking inhalations or bronchial rinsing waters; with negative results - three times examination by flotation method; three times sputum inoculation on nutritive medium, irrespective of the results of the previous examinations, with a view to define MBT sensitivity to antimycobacterial preparations. Further on, in the process of the treatment, the examination is repeated every month until the bacterial secretion ceases, and then once for 2 months till the end of the main course of chemotherapy.
Tuberculin
Robert Koch obtained tuberculin for the first time (1890) which was later named old Koch's tuberculin (Alt Tuberculin Koch - ATK). It is manufactured in ampules as 100 % solution and is a liquid of dark-brown colour, which contains, in addition to specific active substances (tuberculo-proteins), products of MBT vitality, elements of their cells and the medium on which they grew.
In 1934 F. Seibert obtained a more specific (dried) tuberculin preparation - Purified Protein Derivative (PPD-S) - purified tuberculin protein derivate, for which bacteria were grown on a synthetic protein-free medium. In the USSR in 1939 M.A.Linnykova obtained an anological tuberculin preparation, named PPD-L. One ampule contains 50000 TU of dry rectified tuberculin. The solvent is isotonic solution of sodium chloride with the addition of 0.25 % carbolic acid. Preservation time is 5 years, in a dark place at the temperature of + 4° C.
In Ukraine PPD is manufactured as a solution ready for use, the sterility of which is guaranteed by the presence in it of 0.01 % chinozol. The solution is packed in ampules of 3 ml (30 doses) or in bottles of 5 ml (50 doses). Each dose – 0.1 ml contains 2 TU. 0.005 g of twin-80 is added to stabilize biological activity of the solution. In accordance to the WHO international standard 1 TU PPD-L contains 0.00006 mg of dry preparation.
Tuberculin is an incomplete antigen and therefore it does not cause the formation of antibodies, but it calls forth a reaction in a sensibilized organism with a complete antigen (MBT, vaccine strain of BCG).
Depending on the mode of tuberculin introduction, cutaneous Pierquet test (Pierquet, 1907), intracutaneous - Mantoux (Mantoux, 1909) and subcutaneous Koch test (Koch, 1890).
Tuberculin Skin Test
Reactivity to intradermal injection of mycobacterial antigens can differentiate between infected and noninfected individuals. The tuberculin test is a measure of DTH as determined by the intradermal injection of 0.1 ml of intermediate strength purified protein derivative (PPD), which is a tuberculoprotein derived by fractionation of a broth culture filtrate of M. tuberculosis. A positive PPD reaction usually developes within 3 to 4 weeks after exposure. Some infected patients may have less than 10 mm (5-9 mm) induration, but this level of reactivity generally represents exposure to other mycobacteria. Less than 5 mm induration represents negative reaction.
The aim of mass tuberculization:
1. Early tuberculosis revealing.
2. Revealing persons with an increased risk of tuberculosis illness.
3. Contingent selection for BCG re-vaccination.
4. Determination of infestation index of MBT population.
5. Differential diagnostics between infectious and postvaccinal allergy.
The results of Mantoux testing, which are estimated in 72 hours, may be as follows:
1. Negative is absence of a papule or only a sign after an injection to I mm.
2. Doubtful - a 2-4 mm papule or hyperemia only.
3. Positive is a papule of 5 mm or more.
4. Hyperergy - in children and teenagers a papule of 17 mm and more, with adults — 21 mm and more, and also for various age groups, reactions with the availability of vesicules, necrosis or lymphangoitis, irrespective of the papule size.
Individual tuberculin diagnostics.
Depending on the indications at individual tuberculin diagnostics Mantoux test with 2 TU is applied, as well as with various tuberculin doses. Generally, Mantoux test with 2 TU is of importance for children and teenagers; lor adults, in separate occasions, hyperergic results of Mantoux test testify of the active tuberculosis, while the negative ones - of tuberculosis absence, therefore sometimes the necessity arises to apply Koch test (10-100 TU). A negative reaction to 100 TU of tuberculin with the probability of 97-98 % allows to exclude tuberculosis infection. Koch's testing is done with a view of diagnostics and differential diagnostics of tuberculosis, the definition of the activity of tuberculosis process. Before the Koch testing, Mantoux test with 2 TU is done for ascertaining the tuberculin titre. After this near the lower angle of a shoulder blade or in the upper third of the outer surface of the shoulder, after rubbing the skin with 70° ethylic alcohol, tuberculin is injected subcutaneously in the dose from 20 to 100 TU. Two or three days before the procedure the clinical blood analysis is done every day, the intake of lavage waters of bronchi for MBT, measuring the body temperature every 4 hours; a day prior to Koch's test the protein fractions of the blood serum are defined. In 24-48-72 hours after subcutaneous tuberculin injection the examinations analogous to those before the tuberculin injection are done. Roentgenological examination before and after Koch's testing (in 48 hours and on the 7th day) is performed depending on the process localization.
Koch test results
Koch test results are evaluated in 24, 48 and 72 hours, based on the results of local, nidal and general reaction. The local reaction is supposed to be positive at the formation of subcutaneous infiltrate of the 15 mm size and more. The nidal is positive at the availability of the intensification of inflammatory reaction in the site of specific wound. The general (overall) is characterized by the worsening of the general state of the person under examination. It is rising of the body temperature (not less than 0.5°C), joint aches, headache, increased disposition to perspire, as well as changes of the formula and protein fractions of the blood serum (each index, which has deviated not less than 20% from the initial figure is taken into account). Simultaneous changes of not less than 3-4 indices are of diagnostic importance.
Disposition prophylaxis of Tuberculosis
An active vaccine is available that reduces the risk of contracting the disease by about one-half. It contains the live vaccine BCG (lyophilized bovine TB of the Calmette-Guerin type). Vaccination of tuberculin-negative persons induces allergy and (incomplete) immunity that persists for about 5 to 10 years. In countries with low levels of tuberculosis prevalence, the advisory committees on immunization practices no longer recommend vaccination with BCG, either in tuberculin-negative children at high risk or in adults who have been exposed to TB. Preventive chemotherapy of clinically inapparent infections (latent tuberculosis bacterial infection, LTBI) with INH (300 mg/d) over a period of six months has proved effectivity in high-risk persons, e.g., contact persons who therefore became tuberculin-positive, in tuberculin-positive persons with increased susceptibility (immunosuppressive therapy, therapy with corticosteroids, diabetes, alcoholism) and in persons with radiologically confirmed residual tuberculosis. Compliance with the therapeutic regimen is a problem in preventive chemotherapy.
Contraindications for BCG vaccination are:
1) a prematurely born child, when the body mass at birth is less than 2000 g;
2) intrauterine infection;
3) purulent-septic illnesses;
4) hemolytic disease of newly-borns (moderate and severe forms);
5) severe puerperal traumas with neurologic symptomatics;
6) generalized skin wounds;
7) any acute illnesses;
8) generalized BCG infection of other children in the family.
Children (babies), not immunised at a maternity home, in connection with contraindications are vaccinated after recovery at a children's polyclinic or hospital-assistant's health station with BCG-M vaccine during 1-6 months. However, if a baby has reached a 2-months age and more, the Mantoux test with 2 TU should be done before inoculation. Children with negative tuberculine reaction are vaccinated. The interval between Mantoux test and vaccination must be not less than 3 days and not more than 2 weeks.
Morphology and culture properties of Mycobacterium leprae
In morphological terms, these acid-fast rods are identical to tuberculosis bacteria. They differ, however, in that they cannot be grown on nutrient media or in cell cultures.
Epidemiology of Leprosy
Person-to-person spread by direct contact or inhalation of infectious aerosoles. People in close contact with patients who have lepromatous disease are at greatest risk.
Pathogenesis and Immunity of Leprosy
Leprosy manifests as tuberculoid leprosy or lepromatous leprosy, with intermediate forms also recognized. Patients with tuberculoid leprosy have a strong cellular immune reaction but a week humoral antibody response. Infected tissues typically have many lymphocytes and granulomas but relatively few bacilli.
Patients with lepromatous leprosy, however, have a strong antibody response but a specific defect in the cellular response to M. leprae antigens. Thus, an abundance of bacilli are typically observed in dermal macrophages and the Schwann cells of the peripheral nerves. As would be expected, this is the most infectious form of leprosy.
Types of Leprosy
There are several forms of leprosy that range from the mildest indeterminate form to the most severe lepromatous type. More severe forms arise because of less effective immune response to the infection. Most of those infected mount an appropriate immune response and never develop signs of leprosy.
Depending on clinical features, leprosy is classified as:
Indeterminate leprosy (IL)
Tuberculoid leprosy (TT)
Borderline tuberculoid leprosy (BT)
Borderline borderline leprosy (BB)
Borderline lepromatous leprosy (BL)
Lepromatous leprosy (LL)
Laboratory Diagnosis of Leprosy
Microscopic method.
Biological method.
Skin-allergic test with Lepromin.
Treatment of Leprosy
Although at one time the sulfone drug dapsone was the preparation of choice in the treatment of leprosy, the development of a high degree resistance has prompted the recommendation of combined dapsone, rifampin, and clofazimine for patients with lepromatous leprosy and dapsone and rifampin for those with the tuberculoid type.
Addition 2
Fig. 4. The tuberculin skin test is performed to evaluate whether a person has been exposed to tuberculosis. If there has been a prior exposure, sensebilized lymphocytes are formed and remain in the body. During the skin test, the tuberculosis antigen is injected under the skin and if sensebilized lymphocytes are present, the body will have an immune response. There will be an area of inflammation at the site of the injection.
