Bonnet, Maryline

Bonte, L

Camelique, O

Hewison, C

Jones, W

Ruangweerayut, R

Schramm, B

Swaddiwudhipong, W

English

MSF Field Research

Evaluation of the performance and feasibility of the fluorescein diacetate
(FDA) vital staining method for follow up of Tuberculosis (TB) treatment.
Birgit Schramm1, Witaya Swaddiwudhipong2, Ronnatrai Ruengweerayut2, Olivier Camélique3, Laurence Bonte4, Warren Jones5, Cathy Hewison4, Maryline Bonnet1.
1 Epicentre, Geneva, Switzerland, 2 Mae Sot General Hospital, Mae Sot, Thailand, 3 Médecins Sans Frontières, Bangkok, Thailand
4 Médecins Sans Frontières, Paris, France, 5 International Organisation of Migration (IOM), Bangkok, Thailand
Background
TB treatment follow up
Direct Ziehl Neelsen (ZN) smear microscopy: Mycobacterium Tuberculosis (MTB) culture:
 Used in resource-limited settings to monitor TB 
treatment
 Can not distinguish live from dead bacilli
 Literature review: 10-90% of Cat1 or Cat 2 ZN 
smear positive TB failures are culture negative 
 Patients potentially started unnecessarily on a new 
regimen
 Gold standard method 
 Not available in most resource limited settings 
 Long delay for culture results 
Objective
Fluorescein diacetate (FDA) staining distinguishes live and dead bacilli:
live MTB live MTB
FDA
2.  Only in living bacilli:
intracellular hydrolysis (enzymatic activity) 
of  FDA  to   Fluorescein (494 / 518 nm) 
Mycobacterium Tuberculosis 
(MTB)
1.   FDA staining of sputum smear : 
Intracellular uptake of FDA
3.  Detection of life (fluorescent) bacilli by 
fluorescence microscopy
Bangladesh study:
sensitivity = 99%  / specificity= 82 % compared to 
MTB culture when used on fresh sputum 
specimens of TB failures
Hamid Salim A, Aung KJ, Hossain MA, Van Deun A. Early and rapid 
microscopy-based diagnosis of true treatment failure and MDR-TB. 
Int J Tuberc Lung Dis. 2006  Nov;10(11):1248-54
dead MTB
To evaluate the performance and feasibility of the FDA vital staining method compared to MTB culture to 
differentiate live from dead bacilli in ZN smear positive patients during treatment follow-up in a smear 
microscopy laboratory setting
Methods
Study Design: Field evaluation of a TB diagnostic test
Study site: Outpatient chest clinic, Mae Sot, Thailand
Case inclusion criteria: 
 Patient ≥ 15 years 
 Under TB treatment (cat1 or cat2) since ≥ 2 months 
 ≥ 1 of 2 sputum ZN smear positive (≥ 1 acid fast bacilli (AFB) /100 high power fields (HPF)) 
Sputum
day 2
Sputum 
day 1
MTB culture
if  sputum
ZN smear +ve:
Procedure
FDA smear
ZN smear
Staining
1. Air-dried smear from fresh sputum
2. Flood the smear with FDA (0.5 mg /ml)
30 minutes in 37 ºC incubator
3. Rinse with water
4. 1% acid alcohol 3 minutes (de-colorization)
5. Rinse with water
6. 10 minutes 5% watery phenol (safety step)
7. Rinse with water 
8. Dry the smear (30 min -1 hour)
9. Read asap:    
Microscope:
CXC21 Olympus with FRAEN After ® LED Blue 
(480 nm Excitation) / Filter: 535/40 nm bandpass
FDA staining/reading
Reportedresult 1000x magnification Number of fields
Negative 0    AFB   / 100 HPF 100
Scanty 1-9    AFB    / 100 HPF 100
1+ 10-99  AFB  / 100 HPF 100
2+ 1-10  AFB    / 1  HPF 50
3+ >10   AFB   / 1  HPF 20
Sample size and analysis:
218 ZN smear +ve cases
Sensitivity, Specificity, positive and negative predictive values (PPV, NPV):
¾By specimen
¾By case:  
- Positive FDA case: at least 1 out of 2 FDA positive results
- Use of different AFB cut-off (1, 4, and 10 AFB /100  HPF)
to define a positive FDA case
MTB culture, Gold standard
International Organization of Migration (IOM) Laboratory, Mae Sot ,Thailand
• Sputum decontamination (NALC-NaOH-method), spin-concentration
• 3 cultures are inoculated per specimen:
¾1 x Liquid MGIT
¾2 x Solid Lowenstein Jensen
• Species identification: Nitrate Reduction- and Genprobe test
Preliminary Results (continued)
Description of samples according to the stage of TB treatment
AFB count (ZN) versus Culture positivity
Total  Intensive 
phase
Prolonged 
intensive 
phase
Continuation 
phase
End of 
treatment
ZN positive sputum  N (%) 162 (100) 77 (100) 35  (100) 41 (100) 9 (100)
scanty results 126 (77.8) 57   (74) 29  (82.9 ) 33 (80.5) 7 (77.8)
>= 1+  36  (22.2) 20   (26) 6 (17.1) 8 (19.5) 2 (22.2)
Culture positive sputum N (%)  30 (18.5) 22 (28.6)  3  (8.6) 3 (7.3) 2 (22.2) 
among scanty  18 (14.3) 13  (22.8) 2 (6.9) 1 (3) 2 (28.6)
among >= +1   12 (33.3) 9  (45) 1  (16.6) 2 (25) 0
FDA performance:
¾ Per sputum:
%
(95 % CI)
Cut off :
B Culture + Culture  - Total
FDA  +   23 43 66 PPV 34.8 (23.5 –47.6)
FDA - 78 9 96 NPV 92.7 (85.5 –97)
Total 30 132 162
Sensitivity
76.7
(57.7 – 90 )
Specificity
67.4
(58. 7 – 75. 3)
¾ Per follow up-case, using different AFB cut-off:
Operational aspects of FDA vital staining method in a peripheral smear microscopy 
laboratory:
Feasibility
¾ Simple staining procedure, not time consuming (approximately 1.5 hours)
¾ Standard microscope equipped with LED cassette can be used for read-out
¾ Mean reading time for slides = 6.3 minutes (IQR: 5.4, 7.4)
Limitations:
¾ 20°C freezer (storage of FDA stock solution:  25 mg/ml FDA in Aceton)
¾ 37°C incubator (maybe required)
¾ fading of fluorescein signal (within hours):
• Stained smear need to be read shortly after preparation 
• Scanty-loaded AFB-sputum may be miss-read as “fluorescein-negative”
• Challenge for implementation of reliable quality control measures
Discussion
Low FDA performance: possible reasons
Low Sensitivity 
¾ 78% of specimens were ZN scanty (versus 26% Bangladesh study) 
• 6/7 of false negative FDA were on scanty ZN sputum
¾ Study population was not limited to failure cases: Culture positivity 19 % vs 66% in the Bangladesh study
¾ Fading of fluorescence signal
Low Specificity
¾ Poor specificity on fresh specimen (67% versus 82 % specificity in the Bangladesh study)
¾ Scanty live bacilli may be positive in FDA but not in culture 
¾ Preliminary observation: low Fluorescence intensity = risk of false positive ?
Preliminary Results
28th November 2007 - September 2008 MTB culture and FDA results available for:
162 ZN smear +ve sputa 
¾ 89.3 % new cases
¾ 46.4% end of intensive phase
¾ 20.5% prolonged intensive phase
¾ 26.8% continuation phase
¾ 6.3% end of treatment
¾ 48%  cases with 2 ZN smear +ve sputa 
¾ 6.8 % sputum “saliva”
110 ZN smear+ve
follow-up cases 
Considerations
Possibilities for improvement:
¾ Using 2 FDA smear per sputum (sensitivity)
¾ Considering fluorescence brightness at readout ? (specificity) 
¾ Consider AFB cut-off for definition of FDA positive ? (specificity)
¾ Further investigation of FDA performance according to:
• The ZN AFB load
• The stage of TB treatment: ZN-failures versus others 
¾ Further standardization/simplification of the method
¾ Assessment of the method on concentrated specimen
¾ Double staining (fluorescent  markers for “live” versus “dead” in the same specimen),- feasible ?
Acknowledgements
Study team, MSF TB chest clinic, 
Mae Sot, Thailand:
Mr Jadee Tawiwonggkamton
Mrs Kraywa Seangraroengsong
Mrs Paw Lweh Hay Sonklinprai
Mrs Mako Rakarsrom
Mrs Muewapho Kulapkeeree
IOM laboratory, Mae Sot, Thailand:
Mrs Warithorn Madilokkowit
Mr Kittisak Amornpaisarnloet
Damian Foundation, Bangladesh:
Mr M.A. Hossain
Dr A. Hamid Salim
Dr K.A.R. Saki
Mr Subash Chandra Sarker
Mycobacteriology Unit, Institute of Tropical Medicine, 
Antwerpen, Belgium: Dr Armand van Deun Birgit Schramm
Epicentre
Birgit.Schramm@epicentre.msf.org
00 41 (0) 22 849 89 85
MAE SOT GENERAL HOSPITAL
- TAK, THAILAND
Cut off :
Culture + Culture - Total
FDA +   19 30 49 PPV 38.8(25.2 – 53.7)
FDA - 35 8 61 NPV 95.1 (86.3 – 89.9)
Total 22 88 110
Sensitivity
86.3 
(65.1 – 97.1)
Specificity
65.9 
(55 – 75.7)
Cut off :
4 AFB /100 HPF Culture + Culture - Total
FDA +   13 14 27 PPV 48 (28.7 - 68)
FDA - 97 4 83 NPV 89 (80.4-94.9)
Total 22 88 110
Sensitivity
59
(36.6 – 79.3)
Specificity
84.1
(74.5 - 91)
Cut off :
>9 AFB /100 HPF Culture + Culture - Total
FDA +   82 10 PPV 80 (44.4 – 97.5)
FDA - 14 86 100 NPV 86 (77.6 – 92.1)
Total 22 88 110
Sensitivity
36.3
(17.2 – 59.3)
Specificity
97.7
(92 – 99.7)
1 AFB /100 HPF
1 AF / 100 HPF

Evaluation of the performance and feasability of the fluorescein diacetate (FTA) vital staining method for follow up of Tuberculosis (TB) treatment

Evaluation of the performance and feasability of the fluorescein diacetate (FTA) vital staining method for follow up of Tuberculosis (TB) treatment

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