NEED FOR TB DIAGNOSIS
Tuberculosis is a very contagious bacterium which is spread by air as an infected individual coughs or sneezes. In HIV patients, diagnosis is very important before one begins their Anti retro viral therapy because this might make their immune system weak increasing viral load count.
DIAGNOSIS OF TB
When an individual shows signs of tuberculosis they are first tested for HIV in all health facilities in Botswana. When at the health institute seeking for medical assistance the patient is asked for theirmedical, social and duration of symptoms.If the patient has been on treatment this could indicate that the drug is ineffective and medication was not been taken correctly. Common symptoms include prolonged cough, shortness of breath and chest pain and swollen glands. Medical questions that patients are asked include questions like ,if they have been exposed to someone or a family member with Tuberculosis, their history of previous Tuberculosis treatment, their HIV status and history of smoking and drinking. Physical diagnosis then follows. Here, patients may show signs of fever, skin changes and enlarged lymph nodes. Tuberculin skin test are a form of diagnosis. During the Mantoux test, the patient is injected with tuberculin inside the dermis. A small bleb appears after some time. After 48-72 hours the diameter of the bleb is measure and if it less than 5mm the patient is negative of tuberculosis but if it is greater then the individual has tuberculosis. Patients are then taken for an X-ray to examine their lung cavity to check for pulmonary tuberculosis. This is a very sensitive test
TYPE OF SPECIMEN FOR LABORATORY TESTS, THEIR DECONTAMINATION AND LABORATORY SAFETY
The majority of specimens come as sputum. The sputum of an individual is collected into a sterile containerand the container is then labelled with the patients details for easy identification. The sputum can be induced by the inhalation of a hypertonic solution and labelled as induced sputum.There are cases whereby specimens can be received in the form of gastric aspirant in children suspected to have come into contact with Tuberculosis. Anasogastric feeding tube is used to collect the sputum if the child is unable to cough out the sputum. The cerebrospinal fluidcan alsobe collected by creating a lamber puncture at the spinalcord. Tissue can be used which is first centrifudged.The pellet is the grinded and used for further testings.
Decontamination of samples occurs throughout specimen collection and processing to avoid giving false results.N-acetyl 1-Cysteine and sodium hydroxide is used as a digester to remove non-acidcontaminats.To insure the safety of workers, there is a Biosafety level room(BSL3 room)in the facility which is under negative pressure of -40.there is a changing room and the clothing is left inside this room after the manipulation of the bacterium strain.M95 masks are provide which are more reliable than ordinary masks as they prevent the inhalation of the bacterium. In the BSL room, there is a biosafety cabinent.Here, manipulation of samples occurs, sputum specimens from patients opened and bacterial strains are cultured here.The air is this room is continually filtered and sucked
TREATMENT OF SAMPLES
Samples from other health institutes are received by the national Lab in a sealed box to avoid contamination as they are being transported to the lab to determine the External Assurance Quality of the results given to patients. Once all smears have been examined they are disposed of in biohazard bags or if samples are not correct they are resent to the health facility they were taken from so that they are retested again.
Workers are constantly provide with protective clothing including lab coats,N95 masks,gloves.Some rooms such as the master mix room is off limits and only accessible to the staff who work there since it is a very sterile room. In the Biosafety level room no clothing or equipment is brought out after manipulating Tuberculosis samples to avoid outbreaks. The air in the room is constantly being filtered, sucked and cleaned before it is release to the environment.
Examination by mycobacterial culture is the “golden standard” of TB diagnosis. However, culture is expensive and time-consuming than microscopy, and requires the use of specialised media and skilled laboratory personnel to perform the culturing process.The media used is Lowenstein-Jensen (L-J).With this method, a positive result (growth of mycobacteria) is usually apparent after three weeks. If there is no growth by eight weeks, the result is negative for Tuberculosis strain.
Molecular methods for diagnosis Multidrug-resistant Tuberculosis uses the Line probe Assay a form of Polymerase Chain Reaction. These strains are a result of mutation of the Tuberculosis bacterium which changes the DNA sequence of the bacterium .During polymerase chain reaction the following three steps occur being Initiation, Annealing and Elongation. During the initiation steps the two strands are separated at high temperature close to 96oC which are connected by hydrogen bonds. Once the two strands are separates annealing process occurs at temperatures of 42oC to allow primers to attach to the strands. Extension at 72oC of the DNA strand follows. Primers attach and add nucleotides resulting in the synthesis of a new DNA strand.
Some tuberculosis strains are drug resistant as a result, Drug Susceptibility testing occurs. Specimens are collected from sputum of individual’s believed to have a drug resistant Tuberculosis strain or from culture.Isonizide and Rifampicin are the first line drugs used to test for Multi drug Resistant Tuberculosis. Ten fold serial dilutions are carried out. The lowest concentration of 10-3 is used to compare with other serial dilutions. It takes about 28 days to take results.
Staining techniques involves acid fast and Auramine ‘o’ staining .During acid fast staining or Ziehl-Neelsen the following processes occur. Preparation of a smear for staining involves applying a very small sample to the centre of a carefully cleaned glass slide. The microbial sample is usually taken from a broth culture or a suspension of microorganisms produced by mixing a tiny amount of solid matter from colonies with water. The slide is then heat fixed and air dried. The side is Flooded with carbonyl fuchsin and heat until steam rises (3-5 minutes) and then rinsed with tap water .The slide is the decolorized with acid-alcohol for 10-20 seconds.Afterward the Counter stain methylene blue or malachite green is added for 2 minutes.The slide is then Rinsed with tap water, blot dried and observed under the oil immersion objective microscope.There is a possibility that Acid-fast mycobacterium can resistdecolourization by acid-alcohol after primary staining because of the high lipid or mycolic acid content in their cell walls. Therefore the Auramine o staining technique is employed. A smear if first made from a culture suspected of having the bacterium. The slide is then flooded with Auramine O for 15 minutes and thenRinsed with water. Afterwards it is flooded with 3% acid alcohol for 3 minutes followed by rinsing with water and excess water drained from the slide .The slide is then flooded with counterstain potassium permanganate for 30 seconds. Rinse the slide with water; drain excess water from the slide. Phenol is the used to fix the slide. Examinationof the smear is done with a fluorescent microscopewere the bacilli appears brilliant green.