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
 Scratches – oil crystallization
 Spores of bacillus species
o Differentiated by size:
 Mycobacterium – thin
 Spores of Bac. –
boxes
 Transferring elements (“floaters”)
 Fungi
 Stain precipitates
 Exposure of smear/slide to sunlight
 Insufficient reading
 Isoniazid (INH) Antibiotics
 Improper reading (under-reading)
 Too intensive counterstaining
 Improper staining time
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