TB MICROSCOPY DIRECTLY OBSERVED TREATMENT SHORT-COURSE (DOTS) recommended tuberculosis control strategy by the World Health Organization (WHO)

) which is used worldwide developed by Dr. Karel Styblo in 1980s composed of five elements: 1. Political commitment with increased and sustained financing 2. Case detection through quality-assured bacteriology 3. Standardized treatment with supervision and patient support 4. An effective drug supply and management system 5. Monitoring and evaluation system and impact measurement Introduced in the Philippines in 1996 using the Revised National Tuberculosis Control Program (NTP) guidelines NATIONAL TUBERCULOSIS CONTROL PROGRAM (NTP) IN THE PHILIPPINES: Goal : Detect 70% of cases and achieve 80% cure rate Objective : To cut the chain of transmission by detecting and treating infectious TB cases (source of infection) Priority : Sputum smear-positive cases (Active TB) Primary Diagnostic Tool: Direct Sputum Smear Examination DIRECT SPUTUM SMEAR MICROSCOPY (DSSM) Principal diagnostic method adopted by the NTP because: o It provides a definitive diagnosis of active TB o The procedure is simple o Economical o A microscopy center could be put up even in remote areas DSSM Results serve as bases for categorizing TB symptomatics according to standard case definition and are used to : o Monitor progress of patient with sputum smear-positive TB while they are receiving anti-TB treatment; and o Confirm cure at the end of treatment -----------------------------------------------------------------------------------------------------------------------------------------TB Symptomatics any person with cough for two or more weeks with or without the following symptoms: fever, chest and/or back pains not referable to any musculo-skeletal disorders; hemoptysis or recurrent blood-streaked sputum; significant weight loss; and other symptoms, such as sweating, fatigue, body malaise, shortness of breath.

-----------------------------------------------------------------------------------------------------------------------------------------THE PATHOGENESIS OF TUBERCULOSIS: AN OVERVIEW TB is spread from one person to person through the air by droplet nuclei containing TB bacilli (stays in the air for 1 hour) Droplet nuclei containing tubercle bacilli are inhaled, enter the lungs, and travel to the alveoli Tubercle bacilli multiply in the alveoli

A small number of tubercle bacilli enter the bloodstream and spread throughout the body. The bacilli may reach any part of the body, including areas where TB disease is more likely to develop (such as the lungs, kidneys, brain, or bone). Within 2-10 weeks, the immune system produces special immune cells called Macrophages that surround that surround the tubercle bacilli. The cells form a hard shell that keeps the bacilli contained and under control (TB Infection). If the Immune system cannot keep the bacilli under control, the bacilli begin to multiply rapidly (TB disease). As part of the bodys defense mechanism, the body expels (cough out) the TB bacilli from the lungs through the sputum.

ACID FAST BACILLI (AFB) SPUTUM SMEAR EXAMINATION Sputum Collection Sputum - Mucoid material from the lungs brought out by means of coughing which is made-up of 95% fluids (water, mucoprotein) and 5% cellular components (e.g. squamous cells, columnar cells, alveolar macrophage) - Specimen of choice in the bacteriological examination of Pulmonary Tuberculosis Ideal Sputum Specimen o Visual Appearance: Purulent or mucopurulent Cheese-like Yellowish 3 5 ml in quantity o Microscopic Indicators: Presence of at least 25 WBCs/LPO or 4-5 WBCs/OIO Presence of alveolar macrophages or dust cells ---------------------------------------------------------------------------------Alveolar Macrophage/Dust Cell A large, irregularly shaped, phagocytic cell in the alveoli which is mainly responsible in maintaining sterility in the lungs.

--------------------------------------------------------------------------------- 2 Types of Collection According to Purpose of Examination: o Three (3) Sputum Specimens Required from a TB symptomatic to establish a case of TB (For Diagnosis) Should be collected within 2 days Sputum Collection Schedule: Day 1 - SPOT (Random) Collection 1st specimen collected at the time of consultation or as soon as TB symptomatic is identified

Day 2

- EARLY MORNING Collection o 2nd specimen collected in the morning (2-3 hours after wakingup) by the TB symptomatic when he/she is due to submit the specimen to the health facility o Will yield a high percentage of detecting (+) AFB compared to SPOT - SPOT (Random) Collection 3rd specimen collected at the time the TB symptomatic comes back to the health facility to submit the 2nd specimen.

One (1) Sputum Specimen Required from a TB patient to monitor the status of the TB patient after initiation of treatment (For Follow-up) Preferable early morning specimen Sample Sputum Collection Schedule:
Towards the end of nd 2 Mo. Towards the end of rd 3 Mo. Towards the end of th 4 Mo. Towards the end of th 5 Mo. Beginning of th 6 Mo. Beginning of th 7 Mo.

Reg. I Reg. II Reg. III Reg. I

- from the time the patient was diagnosed as smear-positive after the submission of the 3 sputum specimen Reg. II -if smear-positive in 2nd month (follow-up exam), proceed to Reg. II all the way Reg. III - if smear-negative in the 2nd month (follow-up exam) Storage of Sputum Specimens o Should be kept in a cool, dark (away from sunlight) and dry place to avoid liquefaction o Can be stored at room temperature for up to 4 days only from the time of collection o When transport is needed, should be placed in a special box (sputum carrier) and stored in a cool place as possible until it is dispatched (an accompanying Laboratory Request Form which identifies the sputum specimens should be included with each transport box)

Labeling Yearly serial number and order number of sputum specimen on each side of the slide Smear Preparation 1. Fish-out enough yellowish mucopurulent or cheesy particle of the sputum with the flattened rough end of the coco midrib or the end of the wire loop 2. Spread the sputum evenly on a clean labeled glass slide o Standard size of smear is approximately 3cm. in length by 2cm. in width

3. Smear repeatedly in small, circular, coil-like pattern (must be uniform all the time) to allow penetration of the dye into the cell wall of the bacilli during staining. o Smear is considered even if sputum is fully and finely distributed in most of the smear area. o Presence of small circular coil-like pattern of smearing is an indication of an evenly spread smear.

4. Allow the smear to dry at room temperature (Do not dry it under the sun or over a flame) 5. Fix the smear by passing through the flame 2-3 times, about 2-3 seconds. Staining Ziehl-Neelsen Method for AFB Staining Leave enough space between the slides on the staining bridge to prevent the transfer of material from one smear to another. After heating the slide 2-3 times (covered with carbol fuchsin), leave the slide for 5 minutes to allow the waxy part of the bacilli to close. After staining, tilt and air-dry at room temperature using a slide rack (do not expose to sunlight) AFB is distinctly pink or red in color against a blue background Microscopic Examination Smear is of acceptable thickness if the whole depth of smear can be focused sharply, in most fields, under the microscope. Stained smear must be free from stain deposit, dirt, crystals and artifacts, which are commonly caused by overheating of stain and prolonged exposure of the slide to an open environment. Put one drop of immersion oil on the stained smear. Do not touch the smear with the dropper to avoid transfer of AFB from 1 slide to another.

Examine under 1000x objective with 10x eyepiece lens. o Standard Smear Scanning Horizontal Scanning = 150 visual fields

Vertical Scanning = 50 visual fields

Read at least 300 visual fields ( 2 Horizontal line or 6 vertical lines)

Storage of Smeared Slides Dip all examined slides in xylene for 30 minutes to remove immersion oil (separate smearpositive slides from smear-negative slides) Store in the same order of Laboratory register in the slide box Standard Reporting Scale

3+ 2+ 1+ +N 0

PHILIPPINE NATIONAL STANDARD REPORTING SCALE More than 10 AFB/OIO in at least 20 fields 1 10 AFB/OIO in at least 50 fields 10 99 AFB/100 OIO 1 9 AFB/100 OIO (Write actual no. of AFB seen) No AFB seen in 300 OIO

SPUTUM MICROSCOPY EXAMINATION LABORATORY DIAGNOSIS (Based on 3 Sputum Collection) POSITIVE - 2 out of 3 specimens or ALL specimens demonstrate AFB on stained smear NEGATIVE - NONE of the specimens demonstrate AFB on stained smear DOUBTFUL - 1 out of 3 specimens demonstrates AFB on stained smear (Collect another 3 sputum immediately