DIRECT SPUTUM SMEAR MICROSCOPY & ACID-FAST STAINING
Tuberculosis: 6th in the Philippines; 10th in the World
Morbidity: the person is infected with the disease
Mortality: the cause of death
Pulmonary tuberculosis Biosafety Level III
M. tuberculosis – acid-fast bacilli
Urine Specimen – sediments/extrapulmonary (Miliary TB – happens except in the lungs)
Skin (scrapings?); tissue juices – Mycobacterium leprae (ARB positive)
Symptoms: coughing, fever, chills, weight loss
Can progress to: chronic coughing, no appetite
AFB-positive Bacilli – protected by thick waxy coat (mycolic acid)
Can possess MDR (Multi-drug resistance) if left partially untreated
Laboratory Setup
Well ventilated, organized, clean, well-lighted room
Receiving Microscopy Recording Area
Equipment:
Microscope Staining equipment
Biosafety Cabinet Inoculating loop
Slides
Sputum Specimen: comes from lungs excrete by coughing
Ideal specimen:
Mucopurulent – greenish
Cheese-like
Yellowish
TB-DOTS Criteria: blood-streaked is accepted
Bloody sputum, and saliva is rejected
“Pus Cells” – alveolar macrophage (phagocytic cells)
Dust cells – indicative of a good quality smear
Gram staining: >25/LPF epithelial cells rejected
- Squamous cells > pus cells
- Indicative of a salivary components
- Pathogenic organisms are contaminants
>25 Pus cells/LPF = good quality (inversely proportional to epithelial cells)
Collection Time: early morning specimen
3 specimens with at least one Early morning
= spot-morning – spot method
DAY 1: RANDOM DAY 1: TB symptoms
DAY 2: EARLY MORNING 2 visit only DAY 2: Early specimen
DAY 3: RANDOM Random spx. are poor quality DAY 3: TB symptomatic returns
- will yield less rapidly
2 Good Examination are positive of AFB
Another set of specimen: within 1 week; when doubtful of the result
(same collection time.)
Follow-up: during & after treatment (ALWAYS EARLY MORNING)
Significance of a Follow-Up Specimen:
Coarse of the disease
Treatment modification
Treatment outcome
Guidelines on specimen collection
1. Explain clearly
2. What is the good specimen & how to obtain it (educate)
3. …
Patient Collection
Remove any dentures
Avoid mouthwash
o Gargle with water before collection to unwanted remove debris
Storage: cool & dark place
o To avoid liquefaction
o Avoid sunlight, rodents
Transport immediately in a special container
If the sample is need for culture: refrigeration is unadvisable
DSSM
1. Label the slides
2. Spreading
a. 1 loopful of purulent; solid particles
b. >25 pus cells/LPF (“blue dots”)
Test tube layer:
Top layer: saliva layer
o rods/cocci (may be normal flora) with little to none pus cells
Bottom layer: purulent pattern
o 5% saliva; 95% sputum
Dimension:
2x3 cm
o Recommended
1x2 – result to a compact field when viewed
ACID-FAST STAINING
Drying: Heat-fixing (do not boil)
ZN Method: Hot staining
Carbol fuchsin – flooding
Decolorizer – acid alcohol
*Not ideal for cervical, vaginal or urethral spx.
Counterstain – methylene blue
- Malachi green (kinyoun; cold method)
AFB APPLICATION
OIO – examination of cocci/bacilli
o Squamous cell/pus cell examine at LPO/HPO
Proper scanning Improper scanning
or or other variant
Adjacent Viewing Overlapping (1+ result 3+) Under-reading (F.N)
Error in Staining:
Under-staining
Intensive counterstaining
Under-decolorization
AFB-Positive: Pink-red color in a blue background (beaded appearance)
Chinese-character appearance: M. tuberculosis
Nocardia & Corynebacterium may contain mycolic acid
GRADING:
0 NONE
+n 1-9 AFB IN 100 HPF
n = +1 1 AFB
+2 2 AFB’s
... +9 9 AFB’s in 100 HPF
1+ 10-99 AFB/100 HPF
2+ 1-10 AFB/50 VISUAL FIELDS
3+ >10 AFB/20 VISUAL FIELDS
INTERPRETATION:
POSITIVE IF AT LEAST 2 OUT OF 3 SMEARS IS POSITIVE
NEGATIVE 0 OUT OF 3
DOUBTFUL 1 OUT OF 3
Positive slides – 1-2 years storage
False reading: too thick or too thick smear
Factored by the size, thickness, clearness and artifacts
False Positives: False Negatives:
Tap water Exposure of smear/slide to sunlight
Scratches – oil crystallization Insufficient reading
Spores of bacillus species Isoniazid (INH) Antibiotics
o Differentiated by size: Improper reading (under-reading)
Mycobacterium – thin Too intensive counterstaining
Spores of Bac. – Improper staining time
boxes
Transferring elements (“floaters”)
Fungi
Stain precipitates
I. CASE FINDING
Types:
Passive case-finding – voluntary
Active case-finding – consultation, mandatory
Tuberculosis: w/ or w/o x-ray result
Massive hemoptysis
Update: for px’s with confirmatory
Morning-Spot specimen first-morning & spot specimen
Spot-Spot specimen 1 hour apart
Morning specimen: presence/absence of disease
Spot specimen: intensity of disease
II. CASE HOLDING
Pulmonary
Extrapulmonary
Types (“for new px’s”) After treatment:
New Cured
Relapse – cured with positive Completed treatment
Transfer – from other facility Failed
Return after default Defaulted – interruption
Treatment failure Transferred – out
Died
Supervized Treatment – treatment partner (for small positive cases)
TREATMENT PATTERN
Streptomycin (N)
R Rifampicin
I (H)Isoniazid
P Pyrazinamide (Z)
E Ethambutol
CATEGORY I 2 HRZE / 4 HR
CATEGORY II 2 HRZES / 1 HRZE / 5 HRE (for repeat px.)
CATEGORY III 2 HRZ / 4 HR
Cat.I – new
Cat.II - previously treated/failed/defaulted
Cat.III – new smear less severe / extra.
MDR Tuberculosis – treatment with referral to DOTS; NTP coordinator
