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 Médecins 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

Practical aspects  Test reliability  Inter-observer Kappa 0.81 (95% CI )  Test-retest Kappa 0.93 (95% CI )  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