TB diagnosis in peripheral health care centres

Improving direct microscopy by Improving direct microscopy by overnight bleach sedimentation: overnight bleach sedimentation:

a simple tool for peripheral a simple tool for peripheral Health CentresHealth Centres

Maryline BonnetMaryline Bonnet11, Laramie Gagnidze, Laramie Gagnidze11, Willie Githui, Willie Githui22, , Francis VaraineFrancis Varaine33, Andrew Ramsay, Andrew Ramsay4,54,5, Philippe J Guerin, Philippe J Guerin11

11Epicentre, Epicentre, 22Centre for Respiratory Diseases Research, Kenya Medical Centre for Respiratory Diseases Research, Kenya Medical

Research Institute, Research Institute, 33MMédecins Sans Frontières, édecins Sans Frontières, 44Liverpool School of Liverpool School of

Tropical Medicine, Tropical Medicine, 55TDR/WHOTDR/WHO

TB diagnosis in peripheral health care TB diagnosis in peripheral health care centrescentres

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)

RationaleRationale

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

ObjectiveObjective

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

MethodMethod

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)

MethodMethod

Results: Trial profileResults: 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’ characteristicsPatients’ 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 Smear-positive TB patient detectiondetection

Definition of smear-positive patient: > 1 positive smear (N=644)



1 (0.8%) missed case

Smear-positive patient detectionSmear-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



5 (4.2%) missed cases

Smear-positive patient detectionSmear-positive patient detection

Smear-positive specimen Smear-positive specimen detectiondetection

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 aspectsPractical 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

DiscussionDiscussion

Study strengths Prospective and controlled

Outpatient suspected TB cases in peripheral clinic

Standardised concentration method

Practical aspects

Reliability data

Study limitationsAbsence of comparison to the culture Gold Standard

Advantages & disadvantages of the Advantages & disadvantages of the bleach methodbleach 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

ConclusionsConclusions

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

AcknowledgementsAcknowledgements

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 1st 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 1st 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

TB diagnosis in peripheral health care centres

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