Evaluation of a novel concentration method for the microscopic detection of Mycobacterium tuberculosis from induced sputum Pamela Hepple1,2, Philipp du Cros1, Jane Greig1, Soroosh Sereshki3, Reggie Mutetwa4, Leslie Shanks5, Christian Bottomley2, Ruth NcNerney2 1Manson
Unit, MSF, London, UK, 2London School of Hygiene and Tropical Medicine, London, UK, 3MSF OCA Zimbabwe, 4Biomedical Research and Training Institute, Harare, Zimbabwe, 5MSF OCA, Amsterdam, the Netherlands
BACKGROUND
Table 2: Results per TB Beads test version and technique
Diagnosis of tuberculosis (TB) in developing countries relies on sputum smear microscopy, which has poor sensitivity, particularly for patients coinfected with HIV. Induction of sputum by inhalation of saline can increase case detection but requires sample centrifugation. TB Beads (magnetic beads coated with chemical ligands) can be used to concentrate mycobacteria from induced sputum and might be an alternative to complex centrifugation equipment and thus enable increased TB diagnosis in peripheral clinics in areas with high HIV prevalence.
OBJECTIVES Comparison of two methods of induced sputum concentration: routine decontamination and centrifugation, and use of magnetic beads, specific for mycobacteria, to demonstrate the performance of the magnetic beads compared to conventional centrifugation. Implementation of sputum induction for microscopy in a peripheral setting, without routine access to culture, to improve detection of pulmonary tuberculosis in HIV-positive TB suspects.
METHODS In a cross-sectional study, we compared the sensitivity and specificity of TB Beads with centrifugation, against culture on Löwenstein-Jensen medium as a reference standard. Between February and June 2011, all direct smear-negative adult TB suspects presenting to the MSF clinic in Epworth, Zimbabwe were invited to undergo induction, 71% of whom were HIV-positive. Each enrolled patient underwent one induction procedure, inhaling nebulised 6% hypertonic saline for up to 20 minutes. Half of each sample was processed using TB Beads with fluorescent and Ziehl-Neelsen (ZN) microscopy; and half subjected to centrifugation, fluorescent microscopy, and culture on Löwenstein-Jensen medium. The manufacturer changed the formulation of the elution buffer during the study; therefore, results are were analysed by test version, and combined.
Figure 2: Sensitivity, specificity, PPV and NPV of the different microscopy procedures
Figure 1: Algorithm for the processing of induced sputum
DISCUSSION RESULTS
TB Beads showed similar performance on ZN and fluorescent microscopy. TB Beads with fluorescent microscopy had a lower recovery rate than centrifugation with fluorescent microscopy. Löwenstein-Jensen culture did not appear to function as a true reference standard, possibly due to overly-harsh sample decontamination using the Petroff method. While gender did not have an impact on TB Beads results, significantly more men than women were positive on centrifugation (25% of men c.f. 8% of women) and culture (10% of men c.f. 1% of women). HIV status did not significantly affect the results on any technique. 13.1% of patients experienced side-effects as a result of sputum induction. These were mild and could be dealt with on-site and without specific medical intervention.
134 (97%) of 138 patients who gave informed consent produced an induced sputum sample. One sample was misplaced, leaving 133 patients with samples for full analysis. 26 (19%) patients were smear-positive, of whom 21/133 (16%) were positive by centrifugation, and 13 by TB Beads: 9/133 (7%) by ZN and 10/133 (8%) by fluorescent microscopy. Seven patients (5%) were culture-positive and 126 were culture negative. One patient was smear-negative and culture-positive. The sensitivity and specificity of TB Beads microscopy compared with culture were 43% (3/7) and 95% (120/126) respectively for ZN and 43% (3/7) and 94% (119/126) for fluorescent microscopy. CONSTRAINTS When ZN and fluorescent TB Beads microscopy were combined the sensitivity was 57% (4/7). The sample size was not achieved due to slower-than-expected enrolment. However, when centrifugation was used sensitivity increased to 86% (6/7). Smears made from TB Beads eluate were fragile and washed off slides Table 1: Results per microscopy- and/or culture-positive patient easily; this was overcome by prolonged drying and careful staining practices.
CONCLUSIONS Our results show that the TB Beads versions evaluated did not perform well enough to replace centrifugation. Optimised decontamination of induced sputum should be explored. Sputum induction can be successfully and safely conducted in a peripheral setting on ambulant patients. Use of TB Beads in combination with induced sputum and Xpert MTB/RIF ® testing should be explored. Simple and low cost methods are urgently needed for diagnosis of TB in patients co-infected with HIV.
Pamela Hepple Manson Unit, MSF UK pamela.hepple@london.msf.org + 44 (0) 20 7067 4226