Improving direct microscopy by overnight

lameubiquityΜηχανική

21 Φεβ 2014 (πριν από 2 χρόνια και 9 μήνες)

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Improving direct microscopy by overnight
bleach sedimentation: a simple tool for
peripheral Health Centres

Maryline Bonnet
1
, Laramie Gagnidze
1
, Willie Githui
2
, Francis Varaine
3
,
Andrew Ramsay
4,5
, Philippe J Guerin
1


1
Epicentre,
2
Centre for Respiratory Diseases Research, Kenya Medical Research Institute,
3
M
édecins Sans Frontières,
4
Liverpool School of Tropical Medicine,
5
TDR/WHO


TB diagnosis in peripheral health care centres



Ziehl
-
Neelsen Direct Smear microscopy



Only available tool in most settings



50% sensitivity



No new test expected in near future



Improvement of smear microscopy



Sputum collection (e.g. sputum induction)



Sputum processing (e.g. sputum concentration)



Staining and reading (e.g. fluorescence microscopy)

Rationale



Sodium hypochlorite (“household bleach”)
with overnight sedimentation


Encouraging results


Could be implemented in any setting



Still no recommendations due to study
limitations



To evaluate the diagnostic yield and
feasibility of microscopy after overnight bleach
sedimentation in a peripheral laboratory


To compare direct smear microscopy and overnight
bleach sedimentation


Smear
-
positive patient detection


Smear
-
positive specimen detection


To evaluate practical aspects of overnight bleach
sedimentation

Objective



Population


Mathare, Nairobi


High HIV prevalence


>

15 years, pulmonary TB suspects (cough > 2 weeks)



Consecutive sampling



Procedure


Collection of 3 sputa in 2 days


Hot Ziehl Neelsen method

Method


Bleach 3.5%

Specimen
liquifaction with
bleach

Overnight
sedimentation on
the bench

Ziehl Neelsen
microscopy on the
sediment



Case definitions


Smear
-
positive result: > 9 AFB/100 fields


Smear
-
positive patient


>

2 smear
-
positive results


>

1 smear
-
positive result



Analysis


McNemar test for comparison of matched data


Kappa coefficient for inter
-
reader and test
-
retest
reliability (K >0.8: very good reliability)

Method


Results: Trial profile

52 no sputum

Total screened

N= 788

Included

N= 696

Analysed

N= 644 patients

Less than 15 years old =1

Impossibility to produce sputum = 7

Cough < 2 weeks = 20

ICF not signed =63

Other =1

Patients’ characteristics



Mean age: 32.5 years (SD 10.3)



Sex ratio, M/F: 0.8



Past TB history:121 (18.8%)



Intake of antibiotics in the last 2 weeks: 37 (5.7%)



Production of 3 sputa: 614 (95.3%)



Definition of smear
-
positive patient :
>

2 positive
smears (N=621)


121 (19.5%) with bleach method compared to 105
(16.9%) with direct smear method, p=0.02


16 (15.2%) additional patients detected with bleach
method


4 (3.8%) missed cases

Smear
-
positive TB patient detection



Definition of smear
-
positive patient:
>

1 positive
smear (N=644)


138 (21.4%) with bleach method compared to 120
(18.6%) with direct smear method, p=0.001


18 (15.0%) additional patients detected with bleach
method


1 (0.8%) missed case

Smear
-
positive patient detection



Bleach on 2 first sputa versus direct smear
microscopy on 3 sputa (N= 644)


Definition of smear
-
positive patient
>

1 positive smear


133 (20.7%) with bleach method compared to 120
(18.6%) with direct smear method, p=0.01


18 (13.5%) additional patients detected with bleach
method


5 (4.2%) missed cases

Smear
-
positive patient detection

Smear
-
positive specimen detection


Direct smear


Bleach


N= 1879 slides

n

%


n

%

p








>
1 result 1+

301

16.0


363

19.3

<0.001








Non readable slides

3

0.2


36

1.9

<0.001








Aspect
of smear







-

Good

1855

98.7


1610

85.7

<0.001

-

Too thick

5

0.3


5

0.3


-

Too thin

19

1.0


109

5.8


-

Under decolorized

0

0


77

4.1


-

Overheated

0

0


78

4.2










Practical aspects



Test reliability


Inter
-
observer Kappa 0.81 (95% CI 0.71
-
0.85)


Test
-
retest Kappa 0.93 (95% CI 0.89
-
0.95)



Mean duration of bleach sedimentation


Specimen bleaching: 18.6 min


Overnight sedimentation: 16.8 hours



Mean duration of Ziehl
-
Neelsen method


Smearing & drying: 52.9 min versus 21.4 min


Staining: 45.0 min versus 47.1 min


Reading: 3.5 min with both methods

Discussion



Study strengths


Prospective and controlled


Outpatient suspected TB cases in peripheral clinic


Standardised concentration method


Practical aspects


Reliability data



Study limitations

Absence of comparison to the culture Gold Standard

Advantages & disadvantages of the
bleach method



Advantages


Signifiant increase of TB
patient detection


Good reliability


Ease of use


Inexpensive


Bleach
readily available



Disadvantages


Risk of artefacts


Fragile smears


Delay in diagnosis


No standard bleach
quality

Conclusions



Effective, simple and affordable



Further research needed


Best strategy based on cost
-
effectiveness analysis


Feasibility in routine program conditions


Overnight BS and fluorescence microscopy


Shorter sedimentation time

Acknowledgements



Médecins Sans Frontières, study promotor



Stéphanie Charrondière



Tom, Purity, Ali, Andrew and Lucy



The team in Blue House



The team in the Mycobacteriological Laboratory, KEMRI



The Kenyan National TB Control Program

Smear
-
positive TB patient detection


Positive case
detection


DS negative and BS
positive

DS positive and BS
negative


n (%), 95%CI

P

n (%) 95%CI

n (%) 95%CI


BS on 2 1
st
sputa versus DS on 3 consecutive sputa

BS on sputa
1 & 2

133 (20.7) 17.6
-
24.0

DS on sputa 1, 2 & 3

120 (18.6) 15.7
-
21.9

0.01

18/133 (13.5) 8.2
-
20.5

5/120 (4.2) 1.4
-
9.4


BS on the 1
st
sputum versus DS on 2 consecutive sputa

BS on sputum 1

119 (18.5) 15.5
-
21.7

DS on sputum 1 & 2

117 (18.2) 15.3
-
21.4

0.84

13/119 (10.9) 5.9
-
18.0

11/117 (9.4)
4.8
-
16.2