Acid-fast Smear Microscopy Acid-fast Smear Microscopy Details of TechniquesDetails of Techniques

Acid-Fast OrganismsAcid-Fast Organisms

• Primary stain binds cell wall mycolic acids

• Intense decolorization does not release primary stain from the cell wall of AFB

• Color of AFB-based on primary stain• Counterstain provides contrasting

background

Acid-Fast StainsAcid-Fast Stains

• Early diagnosis of mycobacterial infections

• Confirms acid-fast nature of organisms

• Monitor patients on antimycobacterial therapy

• May be a determinant for performing other tests such as culture or susceptibility testing

AFB Microscopy TechniquesAFB Microscopy Techniques

• Two basic techniques: same basic principle– Transmitted light: Carbol fuchsin staining

Contrast red color AFB/ on blue background

– Fluorescence: auramine staining* contrast light/dark;

Basic Fuchsin AFB StainsBasic Fuchsin AFB Stains

• Zeihl Neelsen’s-hot stain

• Kinyoun’s-cold stain

• Modifications

• Most of this discussion will be spent on these techniques

Fluorochrome AFB MicroscopyFluorochrome AFB Microscopy

* Place of fluorescence in low-income ?* more rapid and sensitive* specificity : same with sufficient experience* equipment cost , bulbs, technical demands* for busy labs (> 25 slides daily) * External quality assessment should be done if

this method is performed* Performing EQA PT and rechecking is a

challenge

Fluorescence and Bright-field Fluorescence and Bright-field MicroscopyMicroscopy

Fluorochrome AFB MicroscopyFluorochrome AFB MicroscopyPrimary fluorochrome AFB fluorescesAuramine O GreenAuramine O-Rhodamine B Yellow/orangeAcridine Orange Yellow/orange

Note: Color of AFB may vary with filter system on microscope

Auramine Stain

Fluorochrome AFB MicroscopyFluorochrome AFB Microscopy

Counter Stain Background

Potassium permanganate

Acridine Orange

Dark/black

Yellow/orange

Auramine with Acridine Orange Counter stain

Bright-field TechniquesBright-field Techniques

– Hot Ziehl-Neelsen in practice most reliable* more visible AFB* stronger color

– Cold methods : Kinyoun, Tan Thiam Hok…* less laborious but also less robust* higher concentration fuchsin, longer staining

timeerrors !!

* not recommended for low-income countries

Comparison hot/cold staining, DF Bangladesh

4368 consecutive sputa; 603 positive or scanty by any technique

High False

NegativeLow False Negative

Total False

Negative

High False

PositiveLow False Positive

Quantific. error

Kinyoun 14 47 61 1 4 6

(cold 3% 5 min) 2,3% 7,6% 9,9%

P=0.001

Ziehl-Neelsen 7 25 32 1 3 0

(hot 1%, 10 min) 1,2% 4,1% 5,3%

Multicentre field trial using duplicate smears, with blinding to staining method

Errors defined by rechecking after restaining of all discordant series

Discordance < 1 AFB/ 100 HPF excluded

Sputum SampleSputum Sample

• What is a good sample?– What is saliva? – Good sample = yellow? mucous fluid? – Discharge from the bronchial tree– May contain solid or purulent substances– Minimal amounts of oral/ nasal material– May contain macrophages and other cells

indicative of infectious disease– Follow-up examination samples?

Preparation of AFB SmearsPreparation of AFB Smears

• Smearing– delay: no problem (keep away from sun)– "good particle"

homogenization may be more reliable

– standard size: for ease of quantification

– thickness, evenness: find a balance– sensitivity

light conditions, counter-stain

Sputum Concentration Methods :

Are they worth the effort?

– NaOH digestion + centrifugation : culture

– Hypochlorite (NaOCl) digestion & concentration* homogenization, easy background* oxidation: staining easier; co-flocculation with

proteins ?* concentration by sedimentation, centrifugation, filtration

or flotation

Contradictory reports on efficiency

Bleach Concentration StudiesBleach Concentration StudiesREFERENCE % SM+ / CULT+ % POS. SMEARS

DIRECT BLEACH DIRECT BLEACH

GEBRE 1995, centrif. 31% 69% + 125%

MIORNER 1996, sedim. + 25%

ALLWOOD 1997, centrif. 43% 52% 10.5% 14.8%

WILKINSON 1997, centrif. 43% 44% 12.7% 12.4%

ÄNGEBY 2000, centrif. 57% 65% 9% 12.9%

VAN DEUN 2000, sedim. 15.5% 16.6%

Danger of Cross Contamination

– If tubes re-used – Contamination (water for dilution)

– Need a centrifuge

– Work : specimen preparation or reading ?

• Useful with HIV ?

Fixation of AFB SmearsFixation of AFB Smears

Fixation – may kill some bacilli– makes smear stick to slide– by heat or alcohol– do not overheat– safe smear ?

Primary Staining-ZNPrimary Staining-ZN

Carbol fuchsin staining– Uses higher fuchsin concentration – Dissolve well !!!

IUATLD/WHO : 0.3%

references??

– Heat well-apply long enough – Cold staining!!– Batch staining- great potential for cross-

contamination

Comparison 0.3 vs. 1% fuchsin ZN staining, DF Bangladesh

4626 consecutive sputa; 618 positive or scanty by any technique

High False

NegativeLow False Negative

Total False

Negative

High False

PositiveLow False Positive

Quantific. error

Ziehl-Neelsen 15 42 57 0 7 4

(hot 0.3%, 5 min) 2,4% 6,8% 9,2%

NS

Ziehl-Neelsen 11 40 51 3 5 2

(hot 1%, 5 min) 1,8% 6,5% 8,3%

Multicentre field trial using duplicate smears, with blinding to staining method

Errors defined by rechecking after restaining of all discordant series

Discordance < 1 AFB/ 100 HPF excluded

Decolorization of SmearsDecolorization of Smears

Decolorization– must be complete– not possible to de-stain too much

repeat as needed

– use strong acids– alcohol not absolutely needed

Counter-stainingCounter-staining

– Provides good contrast for observation of AFB

– background for focusing, not too strong– methylene blue 0.3% ?

diluted or < 1 min

use of malachite green?

Microscopic Reading:

Red slender rods on blue backgroundaccept only typical shape, at least some

– depends condition of microscope! light!binocular, mechanical stage, good optics

100x oil immersion objective, 10x eyepieces

– Requires: patience, sincerityAFB microscopy is not difficult but tough

Recording and Interpretation Recording and Interpretation

follow NTP instructions: no. of fields, sputa

– quantification:2 cm = 1 length = 100 HPF– IUATLD/WHO scale

Zeihl Neelsen and Fluorochrome Zeihl Neelsen and Fluorochrome MicroscopyMicroscopy

AFB Quantification ScalesAFB Quantification Scales

System & Quantification Scale

No. of AFB per field

Brightf. & IUATLD/WHO Scale (1000x)

Brightf. & ATS Scale

(1000x)

Fluor. & IUATLD/WHO

Scale

(200-250x)

Fluor. & ATS Scale

(200-250x)

None

1-2/300 fields

1-9/100 fields

1-9/10 fields

1-9/1 field

10-99/1field

>=100/1field

Negative

Actual

Actual

1+

2+

3+

3+

Negative

Actual

1+

2+

3+

4+

4+

Negative

Actual

Actual

Actual

1+

2+

3+

Negative

Actual

Actual

1+

2+

3+

4+