PRESENTATION ON SPUTUM TEST

By Gloria Asantewaa

CONTENT

• Definition of sputum• What is sputum test?• Acid-faststain sputum test• Methodology and results• Limitations and advantages of the test• Precautionary measures• Other tests• Conclusion

OD

WHAT IS SPUTUM?

• Sputum is the liquid substance that is produced from the lower respiratory tract when one coughs.

• In addition to mucus, sputum contains many materials that are not visible to the naked eye. It often consists of bacteria, cellular fragments, blood, and pus.

SPUTUM TEST

• Sputum test is the general test ordered for by a doctor for respiratory tract infections such as pneumonia (caused by Streptococcus pneumoniae) and tuberculosis (caused by Mycobacterium tuberculosis)

• The types of sputum test differ by the types of stain used and the bacteria being tested for.

SPUTUM TEST CONTD.

• The gram stain sputum test is used for detecting gram positive and negative bacteria

• Whiles the acid-fast stain sputum test is used to detect acid fast mycobacteria

ACID-FAST STAIN SPUTUM TEST

• Certain bacterial species like mycobacterium tuberculosis have unusual lipids (mycolic acid) in their cell walls. This substance renders the cell wall very waxy and impenetrable by aqueous stain solutions.

• They therefore have to be stained bythe acid fast stain using carbol fuschin

ACID-FAST STAIN CONTD.

• There are three common acid-fast staining methods, Ziehl-Neelsen (hot), Kinyoun (cold), and Auramine-Rhodamine Fluorochrome (Truant method).

• The slides produced by the Ziehl-Neelson and Kinyoun methods can be visualized using a standard bright-field microscope.

• The fluorochrome method is used by large laboratories that use fluorescent microscopes

ACID-FAST STAIN CONTD.

• In the ‘hot’ Zn technique, the phenol-carbol fuchsin stain is heated to enable the dye to penetrate the waxy mycobacterial cell wall.

• In the ‘cold’ technique known as Kinyoun Method, stain are not heated but the penetration is achieved by increasing concentration of basic fuchsin and phenol and incorporating a ‘wetting agent’ chemical.

ACID-FAST STAIN CONTD.

• The stain binds to the mycolic acid in the mycobacterial cell wall

• . After staining, an acid decolorizing solution is applied. This removes the red dye from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which retain (hold fast to) the dye and are therefore referred to as acid fast bacilli (AFB).

METHODOLOGY

• Collect the sputum sample. • Spread the sputum evenly and thinly over the

central area of the slide with a sterile pippette by a continuous rotational movement. The recommended size of the smear is about 20 mm by 10 mm

• Allow the smear to dry completely. • Fix the smear at 80°C for 15 minutes or for 2

hours on a hot plate set for 65°C to 70°C.

METHODOLOGY CONTD.

• The preferred method of staining(Ziehl Neelsen) is then used.

• Cover the smear with carbolfuchsin stain.• Heat the smear until vapour just begins to rise

(i.e. about 60°C).• Allow the heated stain to remain on the slide

for 5 minutes.• Wash the film in a gentle stream of clean

water until no color appears in the effluent.

METHODOLOGY CONTD.

• Holding the slide with forceps, wash the slide with the decolorizing solvent(3% v/v acid alcohol or 20% sulfuric acid) . Immediately wash with clean water, as above. • Repeat the decolorizing and the washing until

the stained smear appears faintly pink and the fluid washing off the slide runs clear.

• Counterstain with malachite green stain for 1-2 minutes.

METHODOLOGY CONTD.

• Wash well with clean water• Wipe the back of the slide clean, and place it

in a draining rack for smear to air dry.• Examine the smear microscopically, using the

100x oil immersion objective and scan the smear systematically.

METHODOLOGY CONTD.

• Acid-fast bacteria appear red, and non-acid-fast bacteria (and other organisms and cellular materials) appear blue/green.

Mycobacterium tuberculosis viewed under bright field

microscopeA. B.

RESULTS

• When no AFB are seen after examining 300 fields, it is reported as ‘No AFB seen’.(normal)

• When very few AFB are seen i.e. when only 1 or 2 AFB are seen after examining 100 fields, a further specimen is requested to examine (Those AFB might have came from tap water (saprophytic mycobacteria), or it may be scratch of glass slide or by the use of same piece of blotting paper while drying.

RESULTS CONTD.

• When any red bacilli are seen, the smear is reported as‘AFB positive’ (abnormal) and an indication of the number of bacteria present is given as follows:

• More than 10 AFB/field at least in 20 fields: report as + + +

• 1-10 AFB/field at least in 50 fields: report as ++

• 10-99 AFB/ 100 fields: report as +• 1-9 AFB/100 fields: report the exact number

LIMITATIONS OF SPUTUM TEST

• High bacterial load 5,000-10,000 AFB /mL is required for detection

• Smear sensitivity is further reduced in those with extra-pulmonary TB, those with HIV-co-infection, and those with disease due to nontuberculous mycobacteria (NTM).

LIMITATIONS CONTD.

• Limited specificity . All mycobacteria are acid fast .Does not provide species identification.Local prevalence of MTB and NTM determine the predictive values of a positive smear for MTBMicroscopy for acid-fast bacilli (AFB) cannot distinguish Mycobacterium tuberculosis from NTM,Viable from non-viable organisms,Drug-susceptible from drug-resistant strains.

ADVANTAGES OF THE SPUTUM TEST

• Microscopy of sputum smears is simple and inexpensive, quickly detecting infectious cases of pulmonary TB; Sputum specimens from patients with pulmonary TB – especially those with cavitary disease – often contain sufficiently large numbers of acid-fast bacilli to be readily detected by microscopy.

PRECAUTIONARY MEASURES

• Sputum sample should be obtained in a well ventilated area since TB is an airborne disease.

• Sputum sample should be coughed up from deep inside the lungs

• Tap water should not be used in washing off the stains since it may contain saprophytic bacteria that may induce a false positive test

• Smear slide samples should not be blotted dry.

PRECAUTIONARY MEASURES CONTD.

• If the test is not carried out immediately after sample collection, sample may be refrigerated after collection for up to 24hours or kept at room temperature for about 2 hours.

• Sample should be collected early inthe morning after brushing no antiseptic should be used

OTHER TESTS

• There are a number of ways to test for tuberculosis apart from the sputum test. Some are:

• Chest X-ray as a TB test• Culture test• Biopsy• Skin test• Interferon Gamma Release Assay (IGRA)

CONCLUSION

• Checking your sputum is one of the best ways to find out if you have TB disease. If you are already taking medicine for TB, checking your sputum is the best way to tell if the medicine is working.