Optimization of Sputum Smear for Detection of TB: A Prospective Blinded Evaluation Joy Sarojini Michael, Kalaiselvan S, Peter Daley, Lois Armstrong, Shalini E, Poorvi Chordia, Asha Latha, Dilip Mathai, KR John, Madhukar Pai

Introduction Tuberculosis (TB) care, including effective detection and treatment of patients, is central to the global strategy to control the disease Currently available diagnostics are either insensitive, time-consuming, or require laboratory infrastructures Current international guidelines (WHO/IUATLD) recommend the microscopic examination of three serial sputum specimens for acid-fast bacilli (AFB).

 Smear microscopy Disadv : variable and generally low sensitivity (≤ 50%) drop-out of patients during the process but highly specific in high prevelance settings  A systematic review of sputum smear microscopy yielded a mean 18% (95% CI 11-26%) increase in sensitivity when centrifugation was combined with any chemical treatment of sputum samples (Bleach [NaOCl] or lye [NaOH])

Objectives To compare the diagnostic accuracy and incremental yield of two short-duration (<1 hour) sputum pretreatment procedures to direct smears among patients with suspected tuberculosis in India.

Methodology Christian Medical College, Vellore Prospective evaluation over 10 months from Oct 07- July patients Double blinded study Reference standard – culture Lowenstein Jensen media

Inclusion criteria : age ≥ 18 years assessed by a physician who feels the diagnosis of TB is possible willing to consent, HIV test and specimen submission as outlined in protocol Exclusion criteria On ATT within last 6 months Extrapulmonary sample in formalin Unable to produce appropriate specimen

200 TB suspects 11 Extrapulmonary TB suspects excluded 189 pulmonary suspects submitted first sputum for LJ culture 15 did not submit second sputum 175 pulmonary suspects available for smear optimization experiments 239 Patients screened for recruitment 39 not recruited: 10 - Recent TB Treatment 4 - Less than 18 years old 15 - Not available for follow up 3 - Could not produce sputum 3 - Did not fit symptom criteria 4 - No reason given

N% Total pulmonary TB suspects included in smear optimization analysis Male Mean Age40.3 yearsSD 15.8 Mean BMI (N=167)19.6SD 3.97 Outpatient Living in Tamil Nadu state Monthly family income <Rs Experiencing cough Experiencing chest pain Experiencing fever HIV infected148.0 LJ culture of sputum positive Given treatment for TB after recruitment (N=109) Patient demographics

Methodology  4 ml of early morning sputum sample  Homogenised by vortexing  Split into 4 parts Zeihl neelsen Auramine Bleach USP ( universal specimen processing) ZN stain and Auramine fluorescence stain as per standard protocol

Pretreatment of smears  0.5% bleach (NaOCl) + Sputum mixed well distilled water centrifuged smear from deposit  2 volumes USP solution (4M guanidinium HCl, 50 mM tris-Cl, 25 mM EDTA, 0.5% Sarkosyl, 0.1M β-mercaptoethanol, pH 7.5) + Sputum RT for 30 minutes distilled water centrifuge smear from sediment

Number of Acid Fast Bacilli Seen by Staining Method ReportZiehl-Neelsen StainAuramine Stain 1000X200X NegativeZero in 100 HPFZero in 30 HPF Scanty1-9 AFB in 100 HPF (report exact number) 1-29 AFB in 30 HPF AFB in 100 HPF AFB in 30 HPF AFB per HPF (on average) AFB per HPF (on average) 3+>10 AFB per HPF (on average)>100 AFB per HPF (on average)

Results- Incremental yeild of smear optimisation N%95% C.I.P value compared to Direct ZN Smear Total Culture Positives Direct ZN Smear Direct Auramine Smear Bleach Centrifuge Smear USP Centrifuge Smear

Sensitivit y (%) 95% C.I. Specificit y (%) 95% C.I. Positive Predictive Value (%) 95% C.I. Negative Predictiv e Value (%) 95% C.I. Direct ZN smear Direct auramine smear Bleach Centrifuge auramine smear (N=174) USP Centrifuge auramine smear Diagnostic Accuracy of Direct and Pretreated Smears as compared to LJ Culture of Sputum (N=175)

Application In the primary health care setting / DOTS clinics pre treatment can be done to improve sensitivity This can probably also make the sputum less infectious Operational requirements – flurescence microscope & centrifuge needed

Conclusion Pretreatment increased yield marginally by 4% when compared to ZN stain No difference from Direct Auramine stain Small study, needs larger population to study it real benefit in a community setting Pretreatment + Sedimentation technique need to be evaluated

Acknowledgements Departments of Microbiology, Medicine Units & DOTS clinic Christian Medical College Vellore Dr. Madhukar Pai McGill University, Canada

Thank you