# Presentation: 189853

## AFB Smears for Diagnosis and Monitoring Treatment

- Dr. John Ridderhof
- Division of Laboratory Systems
- Public Health Practice Program Office
- Centers for Disease Control and Prevention

## Outline of Presentation

- AFB Microscopy and DOTS Strategy
- AFB Staining Methods / Procedures
- Studies outlining sensitivity and specificity

## TB Control:

**The 5 components of DOTS**

**Political commitment**

**Diagnosis by microscopy**

**Adequate supply of SCC drugs**

**Directly observed treatment**

**Accountability**

## Diagnosis of pulmonary tuberculosis 

- Patients with TB feel ill and seek care promptly
- Active case finding is unnecessary and unproductive
- Microscopy is appropriate technology, indicating infectiousness, risk of death, and priority for treatment
- X-ray is non-specific for TB diagnosis
- Serological and amplification technologies (PCR, etc.) currently of no proven value in TB control

## Diagnosis of Pulmonary Tuberculosis

**Three specimens optimal**
**Spot specimen on first visit; sputum container given to patient**
**Early morning collection by patient on next day**
**Spot specimen during second visit**

## Three sputum smears 

**Three sputum smears **
**are optimal**

## Diagnosis of Pulmonary TB

## X-ray-based evaluation causes over-diagnosis of TB

**X-ray-based evaluation causes over-diagnosis of TB**

- NTI, Ind J Tuberc, 1974

**Over-**
**diagnosis**

## Role of Chest X-ray

- No chest X-ray pattern is absolutely typical of TB
- 10-15% of culture-positive TB patients not diagnosed by X-ray
- 40% of patients diagnosed as having TB on the basis of x-ray alone do not have active TB

- Toman K. Tuberculosis case finding and chemotherapy.  WHO, 1979

- X-ray is unreliable for diagnosing and monitoring treatment of tuberculosis

## Proportion of patients with pulmonary

**TB who have positive AFB smears **

**AFB positivity in **
**TB patients**

## Prompt treatment of infectious cases reduces spread of tuberculosis

- Smear-positive patients usually seek care
- Smear-positive patients are 4-20 times more infectious
- Untreated, a smear-positive patient may infect 10-15 persons/year
- Smear-positive patients are much more likely to die if untreated

- Rouillon A. Tubercle 1976;57:275-99

## DOT prolongs survival of

**HIV-infected TB patients **

**SCC with DOT**

**SCC without DOT**

## Review of main techniques

**Culture**
**more sensitive, but relative**
**low income countries : only 25% gain over smear ?**
**and in these settings less specific ?**
**main problem: DELAYS**
**on classical media (Lowenstein-Jensen....)**
**newer commercial media (BACTEC..)**
**	****	****faster (about 10 days)**
**	****	****but expensive++, technical demands**
**mostly self-healing cases**
**not useful for control in high prevalence**
**first step for susceptibility testing**

## AFB staining

- Ehrlich -1882 discovered the AF nature
- Franz Ziehl-1882
- F. Neelsen in 1883 modified
- Ziehl-Neelsen stain

## Staining methods

**Cold methods : Kinyoun, Tan Thiam Hok**
**Hot method: Ziehl-Neelsen **
**	****	****	****AFB- Red**
**Fluorescence methods: Auramine O- Bright yellow **
**	**** ****Auramine O-Rhodamine B- Yellow orange.**

**Notes:**
Kinyoun: 4g Basic fuchsin
Tan Thiam Hok:basic fuchsine 1 G, 95% alcohol 10 ml, phenol 3 G, distilled water 100 ml ; decolorizing cumcounterstain solution (10% Nacl in water solution 10 ml, Methanol 90 ml + Methylene blue 100 mg) for 30 seconds 
Auramine O 0.1g in 10 ml 95% Ethanol, + phenol 3g in 87 ml of D water
Acid alcohol 0.5 mlHCl in 100ml 70% ethanol.
KMnO4 0.5 g in 100ml of D water. Or Acridine orange can also be used
Staining time: 15, 2, 2 minutes. No heat
 AuraminO- Rhodamine B
1.5G AuramineO + 0.75G Rhodamine B in a solution of 75 ml Glycerol+10ml of melted phenol+50ml of D water.
 Mix well may be clarified by filtering through glass wool.

## Z-N staining method (WHO)

- Primary stain: Carbolfuchsin solution 0.3%
- Decolourising agent: 3% acid alcohol, or 25% Sulphuric acid
- Counterstain: Methylene blue 0.3%

## AFB-MICROSCOPY

- Advantages
- 	-Rapid, high specificity
- 	-Accurate diagnoses
- 		-By paramedical personnel
- 		-Using simple and available equipment
- Disadvantage
-  Low sensitivity

## AFB smear Sensitivity and Specificity

|  | POSITIVECulture | NEGATIVE Culture | TOTAL |  |
| --- | --- | --- | --- | --- |
| POSITIVE
Smear |    A (101) |     C (4) |    105 | PPV  A/ (A+C) |
| NEGATIVE Smear |     B ( 39) |     D (403) |    442
    | NPV D/ (D+B) |
| TOTAL |    140
    |     407
     |    547
    |  |
|  | SensitivityA/ (A+B)          72.1% | SpecificityD/ (D+C)     99.0 % |  |  |

## AFB-MICROSCOPY: SPECIFICITY

- All mycobacterium are acid fast, no exception
- > 98% for AFB
- -In high burden countries
- AFB in sputum = TB
- -In  HIV patients

## SPECIFICITY OF AFB+ SMEARS FOR TB

_**REFERENCE / COUNTRY         HIV+     AFB PATHOGENS, NOT TB**_
**Long, Haiti, 1991****	****      ****	****26%****	****	****4/199 patients**
**	****	****	****	****	****	****	****	****(2/2 HIV+/-)**
**Githui, Kenya, 1992****	****	****33%****	****	****0/320 patients**
**Warndorff, **
**	****Malawi, 1997****	****     ****	****	****>50%?****	****	****2/904 patients**
**	****	****	****	****	****	****	****	****(1/1 HIV+/-)**
**Van Deun, **
**	****Bangladesh, 2001  ****	**** ****	****<1%****	****	****3/1065 patients**

## AFB-MICROSCOPY: SENSITIVITY

- 3 smears = sensitivity of 1 culture
- About 95% of infectious cases
- 		-Priority for treatment
- 		-Control of the disease
- 		-Individual  prognosis

## AFB-MICROSCOPY: SENSITIVITY

- Highly dependent on technical factors
- Quality of sputum, morning sputum 10-100% more positives
- Number of samples
- Quality of smear, staining, quality of microscopes,
- Effort in examining number of fields

## OVERALL SMEAR/CULTURE POSITIVITY RATE
Reference / country                                                            % smear+ out of cult.+  
Boyd and Marr, USA 1975				22 %
Marraro et al., USA 1975				24 %
Rickmann and Moyer, USA 1980			25 %
Urbanczik, Germany					37 %
Burdash et al., USA 1976				43 %
Pollock and Wieman, USA 1977			50 %
MRC, England/Wales 1992				53 %
Kubica, Africa/Europe 1980				53 % (Ziehl Neelsen)
		  , USA/Asia				63 % (fluorescence)
Petersen, Germany					54 %
Blair et al., USA 1976				62 %
Levy et al., S.Africa 1989				66 % (serial sputum)
							54 % (first sputum)
Joseph et al., Indonesia 1976				68 % (fluorescence)
Alausa et al., Nigeria 1977				77 %
Neher et al., Nepal 1997				80 % (3 smears)				(after Urbanczik 1985)
.

_**Reference / country                                                            % smear+ out of cult.+**_**  **
**Boyd and Marr, USA 1975****	****	****	****	****22 %**
**Marraro et al., USA 1975****	****	****	****	****24 %**
**Rickmann and Moyer, USA 1980****	****	****	****25 %**
**Urbanczik, Germany****	****	****	****	****	****37 %**
**Burdash et al., USA 1976****	****	****	****	****43 %**
**Pollock and Wieman, USA 1977****	****	****	****50 %**
**MRC, England/Wales 1992****	****	****	****	****53 %**
**Kubica, Africa/Europe 1980****	****	****	****	****53 % (Ziehl Neelsen)**
**	****	****  ****, USA/Asia****	****	****	****	****63 % (fluorescence)**
**Petersen, Germany****	****	****	****	****	****54 %**
**Blair et al., USA 1976****	****	****	****	****62 %**
**Levy et al., S.Africa 1989****	****	****	****	****66 % (serial sputum)**
**	****	****	****	****	****	****	****54 % (first sputum)**
**Joseph et al., Indonesia 1976****	****	****	****	****68 % (fluorescence)**
**Alausa et al., Nigeria 1977****	****	****	****	****77 %**
**Neher et al., Nepal 1997****	****	****	****	****80 % (3 smears)****	**_**	**__**	**_**	****(after Urbanczik 1985)**
- .

## AFB-MICROSCOPY: SENSITIVITY

**Slightly less in HIV-positives**
**depends on degree of immune-depression**
**also cultures less often positive**
**requires more efforts**
**value of concentration techniques ?**

## RATE OF TB CULTURE+ PATIENTS FOUND SMEAR+
REFERENCE/COUNTRY      TECHNIQUE                    ALL        HIV-        HIV+	
Klein,USA 1989             3-4 sputa, concentr., Kinyoun		81%	45% 
			              	same, first sputum			61%	29%
Long, Haiti 1991	        2-3 morning				79%	66%
Githui, Kenya 1992       3 sputa, fluorescence			89%	89%
Githui, Kenya 1993       ZN staining			65%
			        fluorescence			80%
Elliot, Zambia 1993      3 sputa?, direct, fluor.			76%	57%
Yajko, USA 1994	        1 sputum, concentr., fluor.		55%	55%
Smith, USA 1994	        3 sputa, concentr., fluor.		60%	57%
Allwood, Malawi 1997  1 smear, ZN, direct		43%
Finch, USA 1997	        many sputa, fluor.			71%	70%
			        first sputum				64%	55%
Johnson, Uganda 1998					98%	96%
Karstaedt, S. Africa 1998					79%	68%		

_**REFERENCE/COUNTRY      TECHNIQUE                    ALL        HIV-        HIV+**__**	**_
**Klein,USA 1989             3-4 sputa, concentr., Kinyoun****	****	****81%****	****45% **
**	****	****	****              ****	****same, first sputum****	****	****	****61%****	****29%**
**Long, Haiti 1991****	****        2-3 morning****	****	****	****	****79%****	****66%**
**Githui, Kenya 1992       3 sputa, fluorescence****	****	****	****89%****	****89%**
**Githui, Kenya 1993       ZN staining****	****	****	****65%**
**	****	****	****        ****fluorescence****	****	****	****80%**
**Elliot, Zambia 1993      3 sputa?, direct, fluor.****	****	****	****76%****	****57%**
**Yajko, USA 1994****	****        1 sputum, concentr., fluor.****	****	****55%****	****55%**
**Smith, USA 1994****	****        3 sputa, concentr., fluor.****	****	****60%****	****57%**
**Allwood, Malawi 1997  1 smear, ZN, direct****	****	****43%**
**Finch, USA 1997****	****        many sputa, fluor.****	****	****	****71%****	****70%**
**	****	****	****        ****first sputum****	****	****	****	****64%****	****55%**
**Johnson, Uganda 1998****	****	****	****	****	****98%****	****96%**
**Karstaedt, S. Africa 1998****	****	****	****	****	****79%****	****68%****	**_**	**_

## AFB MICROSCOPY TECHNIQUES

- Sputum decontamination and concentration methods :
- Smear: Hypochlorite (NaOCl) digestion
- Culture: NaOH digestion, NALC digestion Danger of carry-over (reused tubes), contamination (water for dilution) and requires centrifuge

## POSITIVITY RATES IN AFB FOLLOW-UP 

**POSITIVITY RATES IN AFB FOLLOW-UP **

**EXAMINATIONS, KINSHASA 2000**

**0**

**2**

**4**

**6**

**8**

**10**

**12**

**14**

**16**

**18**

**0**

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**11**

**12**

**13**

**14**

**15**

**16**

**17**

**18**

**19**

**20**

**% POSITIVE FOLLOW-UP SPUTUM SMEARS**

**NUMBER OF CENTRES**

**POOR MICROSCOPES**

**POOR QUALITY CONTROL**

## POSITIVITY RATES IN AFB FOLLOW-UP 

**POSITIVITY RATES IN AFB FOLLOW-UP **

**EXAMINATIONS, DF BANGLADESH 1999**

**0**

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**0**

**1**

**2**

**3**

**4**

**5**

**6**

**7**

**8**

**9**

**10**

**11**

**12**

**13**

**14**

**15**

**16**

**17**

**18**

**19**

**20**

**21**

**22**

**% POSITIVE FOLLOW-UP SPUTUM SMEARS**

**NUMBER OF CENTRES**

**GOOD MICROSCOPES & STAINS**

**REGULAR EFFICIENT QUALITY ASSURANCE**

## Thank You