Guidelines on Standard Operating Procedures for MICROBIOLOGY

Chapter 17 - Tuberculosis

Tuberculosis is caused by Mycobacterium tuberculosis which is an acid fast bacillus (AFB). The highest priority for tuberculosis (TB) control is the identification and cure of infectious cases i.e. patients with sputum smear positive pulmonary TB. The highest priority in the diagnosis of TB is thus given to sputum microscopy. The culture of Mycobacterium tuberculosis may only be feasible at a few of the intermediate laboratories.

     Morphology of M.tuberculosis    

Acid fast bacilli are approximately 1-10 m long, slender, rod-shaped bacilli which may be curved or bent. These may be granular, isolated, in pairs or in groups. Stained bacilli may present a beaded appearance.

     Microscopy of sputum

Diagnosis of pulmonary TB by sputum microscopy is simple, easy, inexpensive, rapid, technically not very demanding and more reliable than X-rays. The purpose of the sputum microscopy is two fold (a) diagnosis of the patients with infectious tuberculosis (b) monitoring the progress. For diagnosis, 3 sputum examinations are performed (spot, morning, spot) and for follow up 2 sputum examinations (morning, spot) are performed.

     Collection of sputum sample

     Select a good wide-mouthed sputum container, which is disposable, made of clear thin plastic, unbreakable and leak proof material.

     Give the patient a sputum container with the laboratory serial No. written on it. Show the patient how to open and close the container and explain the importance of not rubbing off the number written on the side of the container.

     Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and spit in the sputum container by bringing it closer to mouth.

     Make sure the sputum sample is of good quality. A good sputum sample is thick, purulent and sufficient in amount (2-3 ml).

Give the patient another container with laboratory serial number written on it for an early morning specimen. Explain to the patient to rinse his/her mouth with plain water before bringing up the sputum.

     Storage and transportation of specimens

If the specimen is collected in the field and cannot be immediately processed, it should be transported to the laboratory within 3-4 days of collection. The specimen should be collected in the containers meant for the purpose, lid tightly secured, properly labelled and kept away from the sun and heat. These can be placed in a special box which can withstand leakage of contents, shocks and other conditions incident to ordinary handling practices. These boxes should be kept in the cooler conditions and then transported to the laboratory.

      Preparation of smear and Ziehl Neelsen staining (AFB staining)

     Select new unscratched slide and label the slide with Laboratory Serial number.

     Make a smear from yellow purulent portion of the sputum using a bamboo stick. A good smear is spread evenly, 2 cm x 3 cm in size and is neither too thick nor too thin. The optimum thickness of the smear can be assessed by placing the smear on a printed matter and the print should be readable through the smear.

     Let the smear air dry for 15-30 minutes.

     Fix the smear by passing the slide over the flame 3-5 times for 3-4 seconds each time.

     Stain the smear by Ziehl Neelsen method as described in Chapter 4.

     Grading of microscopy smears

Record the results in laboratory form and laboratory register appropriately as per table given below:

Store all positive and negative slides till instructed by supervisor to destroy them.

      How to prevent false positive sputum results?

     Only use new, unscratched slides.

     Always use filtered carbol fuchsin.

     Do not allow the carbol fuchsin to dry during staining.

     Do not allow the carbol fuchsin to boil during staining.

     Decolorize adequately with sulphuric acid.

     Make sure there are no food particles or fibres in the sputum sample.

     Never allow the oil immersion applicator to touch a slide.

     Never allow the oil immersion lens to touch a slide.

     Label sputum containers, slides, and laboratory forms accurately.

     Record and report results accurately.

      How to prevent false negative sputum results

     Make sure the sample contains sputum and not just saliva.

     Make sure there is enough sputum (at least 2 ml).

     Select thick, purulent portions to make the smear.

     Prepare smears correctly – not too thick, too thin or with too little material.

     Fix for the correct length of time, not too short or too long.

     Stain with carbol fuchsin for 5-7 minutes.

     Do not decolorize with sulphuric acid too intensively.

     Examine every smear for at least five minutes observing at least 100 fields before recording as negative.

     Label the sputum containers, slides and laboratory forms carefully.

     Record and report results accurately.

     Culture for M. tuberculosis   

     Indications  

Each and every case of suspected tuberculosis need not be cultured. The judicious use of culture limits its use to the followings:

     Diagnosis of smear negative pulmonary TB cases with strong clinical and radiological suspicion.

     Diagnosis of extrapulmonary TB.

     Follow up of a case to investigate for a drug resistant isolate.

     Diagnosis of childhood tuberculosis.

     Procedure  

In some of the intermediate laboratories, the culture for M. tuberculosis may be feasible – wherever indicated and possible. The procedure recommended is given below:

     In a small glass, sealable bottle, mix equal volumes of sputum and 4% sodium hydroxide.

     Shake well and incubate at room temperature (25-30oC) for 15-20 minutes with regular shaking every 5 minutes.

     Centrifuge at high speed (>13,000 g) for 8-10 mts.

     Discard the supernatant.

     Neutralize the sediment by adding drop by drop, 2 mol/litre HCl containing 20 ml of phenol red solution per litre until the mixture remains pink.

     Inoculate the neutralized deposit on to atleast 3 tubes of Lowenstein-Jensen (L-J) medium-one with glycerol and another with sodium pyruvate.

     Incubate the L-J tubes for 2-3 days at 35-37o C in a horizontal position with the caps loosened half a turn. Thereafter incubate at 37o C for 6 weeks and inspect for growth at weekly intervals. Initial incubation of the culture tubes in the presence of 5-10 percent CO2 improves the growth of M.tuberculosis.

     Note the growth of bacteria on the surface during these weekly inspections, and if present stain by Ziehl-Neelsen method.

     If the isolate has the typical colonial appearance and the Z-N stained smear from a colony is also typical report the growth as Mycobacterium spp.

     Send the isolate to a reference laboratory for further characterisation and susceptibility testing.

      Biosafety

     Treat all sputum samples as potentially infectious and use leak proof containers for collection and transportation.

     Use bacteriological safety hood while carrying out all procedures involving sputum.

     Dispose of the sputum cups by incineration, autoclaving or treating with 5% phenol or 2% freshly prepared hypochlorite solution, whichever is feasible.

     Take special precautions while transporting the cultures to the reference laboratories with emphasis on containers which don’t get broken in the event of an accident.

     Wash hands with soap and water frequently especially after touching the sputum.

     Do not eat, drink or smoke in the laboratory area.

     Clean laboratory bench tops with a disinfectant (phenol or hypochlorite solution) at the end of each day.

     Quality assurance  

     All sputum smear positive slides and 10 percent of the randomly chosen negative slides be examined by the laboratory supervisor. There should be high degree of concordance between the results reported in the initial reading and that by the supervisor reading. If the persistence of false positive and false negative results is noticed, review all the analytical and para analytical factors.

     Quality assurance must be applied to safe laboratory arrangement, equipment, collection and transportation of specimen, reagents and methods and reporting of results. Use standard operating procedures, equipment should meet the manufacturer’s claims and specifications and reagents should be of good quality. Analyze laboratory results on a weekly or monthly basis.Adequately trained and motivated staff is crucial to the generation of quality results.        

     Susceptibility to antituberculosis drugs

There is no need of undertaking susceptibility testing for anti-TB drugs as a routine. The resistance in tuberculosis has been found to be mainly due to noncompliance of treatment,rather than due to drug resistant bacilli. Moreover, the second line of drugs is very toxic, expensive and less effective than the first-line drugs. Anti-TB susceptibility testing is technically very demanding and should be done only in some selected reference laboratories.

      Reporting of results

      Sputum microscopy

     If all three specimens or two specimens show presence of AFBs: Report positive.

     If one of three specimens show AFB, correlate the findings clinically and radiologically.

     If none of the three specimens shows AFBs: Report negative.

     Positive culture

Report positive when typical colonies are isolated and Ziehl Neelsen staining shows acid fast bacilli.

     Negative culture

If no growth is obtained after 6-8 weeks of incubation report negative.

      Referral

     As a quality assurance procedure

     For further characterization and storage of isolate

     Susceptibility testing in cases of treatment failure/relapses

     Further reading      

1.      CH Collins, JM Grange, MD Yates: Organization and practice in tuberculosis bacteriology, Butterworths, 1985.

2.      M Salfinger, GE Pfyffer. The new diagnostic mycobacteriology laboratory. Eur J Clin Microbiol Infect Dis 961-977, 1994.

3.      AD Harris, D Maher, P Nunn Practical and affordable measures for the protection of health care workers for tuberculosis in low income countries. Bull WHO 75:477-489,1997.

4.      DA Enarson, HL Reider, T Arnadottier, A Truebucq Tuberculosis guide for low income countries. IUTLD, Paris France, 1996.