SPUTUM AFB TEST

Demonstration of AFB by sputum microscopy is the most confirmatory test for pulmonary tuberculosis, but one has to be sure that sputum has come from lungs and it is not just saliva.

Single direct smear miss about 25% of sputum positive cases

Simple, inexpensive, appropriate technology which is relatively easy to perform and to read, yields timely results with very high sensitivity of detection of tubercle bacilli.

Early diagnosis of mycobacterial infections.Confirm acid fast nature of organism.Monitor effectiveness of treatment.May be determinant of performing other tests

such as culture or susceptibility testing.

INDICATIONS

Severe cough for 2 or more weeks (often involving blood loss)

Weight lossNight sweatsWeakness or fatigue(tiredness)ChillsFeverPain in the area infected.

SYMPTOMS OF TUBERCULOSIS

Under NTP conditions, the IUATLD recommends collecting 2 sputum samples “on the SPOT-early MORNING” preferably within 2 days, from each person presenting at health centre, with respiratory symptoms of more than 3 weeks duration.

Volume of sputum: 3ml-6ml(minimum acceptable amount:2ml)

SPOT specimen when TB suspects present at the health centre.

MORNING specimen sputum produced within 1 or 2 hours after rising.

SPUTUM COLLECTION

If sputum are collected within the same 24 hour period, a minimum of 8 hours between specimen is required.

Sputum smear positive tuberculosis a person presenting with respiratory symptoms with at least one positive sputum smear microscopy examination.

Detects about 80% of TB suspects ultimately positive on sputum smear examination with the 1st specimen, and additional 15% with the 2nd specimen .

5-10 mlThick and mucous, but may be fluid with

pieces of purulent material.Colour varies from opaque white to green,

but reddish to brown when blood is present.

CHARACTERISTICS OF A GOOD SPUTUM SPECIMEN

Primary stain binds cell wall mycolic acid.Intense decolorization does not release the

primary stain from the cell wall.Colour of AFB based on primary stain.Counter stain provides contrasting

background.

ACID FAST ORGANISMS

Transmitted light: Carbol fuchsin staining.[contrast red colour AFB on blue background]

Fluorescence : Auramine staining[contrast light/dark]

AFB MICROSCOPY TECHNIQUE

PROCEDUREMake the smear on a clean glass slide, air dry and

keep it fixed.Add conc. Carbol fuchsin, kept for 5-7 minutes,

with intermittent heatings (till fumes arise), wash with water.

Add 20% H2SO4, kept for 5 minutes, wash with water.

Add methylene blue, kept for 3 minutes, wash with water.

Air dry and observe under oil immersion objective of microscope.

ZN(ZIEHL NEELSEN) STAINING

ZN STAINING

ZN STAINING

AURAMINE STAINING

A well stained ZN smear shows strong red AFB against a weak blue background.

Examine 100 fields with the bright field microscope, 1000X magnification.

Declare the smear negative if no AFB are found.

For high positives examination of 20-30 fields may suffice.

EXAMINATION OF ZN SMEAR

No.

Examination(No. of bacilli)

No. of fields Grading

1 0 300 -ve

2 2-3 300 Doubtful, repeat smear.

3 1-9 100 1+

4 1-9 10 2+

5 1-9 1 3+

6 >10 1 4+

GRADING OF AFB SMEARS

Acid fast particles and other micro-organisms(waxes, precipitate of stain, nocardia, environmental mycobacteria, spores of bacillus subtilis, fibres and pollens, food particles, yeast and artifacts like scratch on slide)

FALSE POSITIVE

Inadequate sputum collection, failure to select suitable sputum particles for making smear, poor smear preparation and inadequate examination of smears.

FALSE NEGATIVE

Specific and confirmatory test for pulmonary tuberculosis.

It is the test for infectiousness of the patient.Sputum examination is important to monitor

progress during chemotherapy.It helps to detect drug failure and

development of resistance in patients in anti tubercular treatment(fall and rise phenomenon)

It is most cost effective investigation.

ADVANTAGES

It is less sensitive investigation compared to radiology. Since number of bacilli required for demonstration of AFB in sputum is quite enormous(10,000 bacilli/ml3), the cases excreting less number of bacilli may not be diagnosed by microscopy alone. Similarly cases of pleural effusion and hilar lymphadenitis may also be missed.

Does not differentiate between living and dead bacilli.

Typing of bacilli is not possible.False positive result can mislead in diagnosis.

DISADVANTAGES(when used alone for diagnosis)

Sensitivity can be increased by concentration method, processing sample with NALC-NAOH method require expertise, but can detect 15-20% more cases.