3.4 Laboratory methods of mycobacterium tuberculosis diagnosis


In the future it will not be difficult to decide what tuberculosis is and what is not… The demonstration of tubercle bacilli… will settle the question

Robert Koch

Laboratories serve major roles in the diagnosis and management of tuberculosis – to reveal MBT at a patient. The laboratory MBT identification consists of the following methods:

  1. sputum collection and processing;
  2. microscopic identification of MBT in secretions or tissues;
  3. cultivation techniques;
  4. drug susceptibility testing;
  5. serological testing;
  6. performance of novel molecular bi¬ological methods of MBT identification, including polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP).

Collection of sputum specimens with MBT could be made in special medical institutions and in various types of outpatient clinics. The collected specimens should immediately be sent to the laboratory for examination. Special sputum containers should be used. They must be rigid to avoid crushing in transit and possess a wide-mouthed screw top hermetically scalable cover to prevent desiccation and to minimize contamination by leakage. There are two acceptable types of container. One available from UNICEF (UN Children’s fund), is plastic with a black bottom, a translucent lid, and is readily destroyed by burning; the patient’s identification must be made on the container (not on the lid). The other is a heavy glass, screw-capped jar that may be re-used after disinfections by boiling (10 min.) and thorough cleaning.

There is a very high risk of infection when the patient coughs, therefore specimens should be collected as far away as possible from other people in specially prepared room.

Additional procedures for MBT collection.

Laryngeal swabs.
The operator should wear a mask and gown when taking a swab: the patient’s tongue is held using a piece of lint and the swab pushed down behind the tongue towards the larynx. The patient will cough and the swab will catch some mucus. Put the swab back into the sterile bottle and send to the laboratory for culture.

Bronchial flush waters.
For good time diagnostics of lung tuberculosis and tracheal and bronchial systems the large importance has early recognition of the bronchi defeat. The bronchi flush waters examinations are applied in practice for this purpose. The technique of bronchi flush waters is not difficult, but it is necessary to remember contra-indications of its application. This technique should be applied with large care to the people of senile age. The procedure is contra-indicated at bronchial asthma, and also at the signs of heart-lung insufficiency.

For reception of bronchial flush waters first anesthesia is carried out of patient’s upper respiratory ways. With the help of the larynx syringe the 15-20 ml of a physiological solution, are released into, warmed up to 37 C. It strengthens secretion of bronchial mucosal expectorations. The patient coughing out expectorates flush waters. The waters are collected to sterile flasks and are processed by the usual way for bacterioscopy or for MBT cultivation. The examinations could be made in separate bronchus or for the whole branch. The method of bacterioscopy of bronchial flash waters and especially their cultivation on media has increased number of MBT reveals on 11-20 %.

Gastric suction (Gastric flush waters).
Gastric suction usually used among children who are not able to cough out sputum, and also at those adults, which have poor amount of sputum. The method is not difficult and gives rather high percent of detection MBT not only with lung tuberculosis, but also with tuberculosis of other organs (skin, bones, joints etc.). For reception of gastric flush waters the patient in the morning should have to drink a glass of water. Then through gastric tube collect waters from the stomach in sterile flasks. After that, water is centrifuged, and then smear is produced from purulent elements of the received precipitate and is stained by a usual way, as the sputum.

Examination of cerebrospinal fluid.
At suspicion of tubercular meningitis it is necessary at the first days to make the analysis of cerebrospinal fluid. When the cerebrospinal fluid is obtained, attention should be paid to a degree of pressure, under which it is discharged from the channel. The liquid flowing by a continuous jet under the large pressure, testifies about increased intracranial pressure. If the liquid expectorates by large often drops, it is a sign of normal pressure, and rare small drops – about the lowered pressure or about of the obstacles of it discharges. Material for research is placed in two sterile tubes. One tube is left in the cold and after 12-24 hours the film is formed on the surface of the liquor. From another tube the liquor is send for biochemical and cytological examinations.

When other methods have failed to make a diag¬nosis it is possible to collect bronchial material by a trap specimen through a bronchoscope. Biopsy of the lining of the bronchi may some¬times show typical changes of tuberculosis when hystologicaly examined.
Pleural fluid.
MBT may occasionally be seen in centrifuged pleural fluid, but usually are only found on culture. The larger the amount of fluid cultured the more likely it is that you will get a positive result.
Pleural biopsy.
Pleural biopsy can be useful when there is pleural effusion. But it needs a special biopsy needle (Abrams punch), facilities for histology and training in the technique.
Lung biopsy.
Only experienced surgeons should use this method. A diagnosis may be made by histology or by finding ТВ in the sections.

Sputum microscopy.

For more than 100 years exists the most simple and fast method of revealing acid fast bacilli (AFB) is sputum microscopy. AFB –mycobacteria that stay stained even after they have been washed in an acid solution and may be detected under a microscope in a stained smear. Mycobacterium differs from other microorganisms by characteristic structure of their cell wall consisting from mycolic acids, which, due to sorption properties, cause ability to be painted on techniques revealing an AFB. This resistance to standard dyes and the avidity with which the mycobacterium retain certain dyes, once impregnated, are both due to the high lipid content of their cell walls. Generally, gram-positive bacteria have about 5% lipid or wax content in their cell walls, gram-negative organisms around 20%, and the mycobacterium roughly 60%. Sputum mycroscopy or other separated material with MBT is carried out by a “simple” method and method of flotation. In simple method, smear is prepared from sputum lumps or drops from liquid substance (exudates, flash waters etc.). A material is placed between two subject glasses. One of smears is processed be Gram’s stain, for revealing common flora, another – for MTB reveal.

Techniques that are still employed today-the carbol-fuchsin methods of Ziehl-Nielsen. The fundamental principles of these methods relate to the ability of the cell wall to absorb carbol-fuchsin dye. The mycobacterial cell walls absorbing the red carbol-fuchsin are becoming so impregnated, that they resist decolorization even with a potent hydrochloric acid-ethanol solution (acid-alcohol). So, when the slide is counterstained with methylene blue, the mycobacterium appears as a red rod on the blue background.

Since 1989, fluorescence microscopy has largely supplanted the older, acid-fast methods in modern laboratories. Fundamentally, this technology relies on the same lipid-related avidity for dyes, but the stain in this case is auramine-rhodamine. The mycobacteria absorb this agent and resist decolorization with acid-alcohol; however, the auramine-rhodamine-stained bacilli fluoresce when excited with UV or other specially filtered light frequencies. The mycobacteria appear as bright yellow rods against an inky black background in the UV system.

Preparation of the sputum for cultural examination.

When diagnostic material with the probable MBT contents is accepted in modern microbiological laboratory the following diagnostic manipulations will be carried out.

  1. Mycolytic acids (MAC) are added to disperse protein substances.
  2. A sample decontamination to eliminate the normal respiratory flora.
  3. Shaking of the mixture and to stand for precipitation.
  4. Centrifugation in a refrigerator.
  5. Pellet containing most of the bacilli collects in apex of tube used for microscopy and culture plating inoculation
    1. Lowenstein-Jensen inspissated egg;
    2. 7-11 Selective agar;
    3. Automated liquid culture systems (MB/BacT или BACTEC MGIT 960).

MBT molecular-genetic diagnostic methods.

Deciphered MBT genome has opened unlimited prospects, in development of the genetic-molecular tests, including in study and revealing MBT and its diagnostics in the host. The classical methods used for detection of MBT presence in host, such as bacterioscopy, cultural, serological, cytological are rather effective, but differ either insufficient sensitivity, or duration of revealing МВТ. Development and the perfection of molecular-diagnostic methods has opened new prospects for fast revealing MBT in clinical samples.

Polymerase Chain Reaction is mostly used (PCR). Polymerase chain reaction entails amplification of characteristic fragments of bacillary DNA that is found in diagnostic specimens It is to be used for sputum specimens that are smear positive or to identify species that are growing in culture medium. The PCR allows to carry out MBT identification in a diagnostic material for 5-6 hours (including processing of a material) and has high specificity and sensitivity (in a range from 1-10 of cells in a sample).

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