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  1. Richard N van Zyl-Smit1,
  2. Richard Meldau1,
  3. Keertan Dheda1,2,3
  1. 1Lung Infection and Immunity Unit, Division of Pulmonology and UCT Lung Institute, Department of Medicine, University of Cape Town, South Africa
  2. 2Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, South Africa
  3. 3Centre for Infectious Diseases and International Health, Department of Infection, UCL Medical School, London, UK
  1. Correspondence to Richard N van Zyl-Smit, J Floor OMB Groote Schuur Hospital, University of Cape Town, 7925 Observatory, Cape Town, South Africa; rvzs{at}

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We thank Lange and colleagues for their insightful comments about our data.1 In our study, one-third of the bronchoalveolar lavage (BAL) enzyme-linked immunospot (ELISpot) test results were indeterminate.1 Lange et al pose the question of whether the number of indeterminate results could be reduced by redefining the cut-off point used for the analysis.

There were 28/83 indeterminate results (33.7%), of which less than half (13/28 or 46.4%) were due to high spot counts in the negative control well. When we reanalysed the data with the cut-off point used by Lange and colleagues,2 four additional subjects had valid results. On reanalysis the sensitivity remained unchanged and the specificity was marginally reduced from 93.75% (95% CI 79.85 to 98.27) to 91.67% (95% CI 78.17 to 97.13). Many of the high spot counts in the negative control well were not close to the cut-off point. In our original analysis we were not able to reduce the number of indeterminate results without significantly compromising the sensitivity when changing the cut-off point of the negative control. Furthermore, in most cases there was little difference between the counts in the negative control and antigen-specific wells, suggesting an effect of terminally differentiated effector cells rather than one attributed to antigen-specific cells.

Nevertheless, we found that 53.6% (15/28) of our indeterminate results were due to failure of the positive control. We showed that using staphylococcal enterotoxin B (SEB), in addition to phytohaemagglutinin (PHA), substantially reduced failure of the positive control (25–3%; p=0.02). We estimate that if SEB was used as a positive control throughout the study then the proportion of inconclusive RD-1 ELISpot results would have dropped from 34% to 25%. We therefore recommend that SEB and PHA be used as positive controls in the BAL ELISpot assay.

In addition to the selection of cut-off points, the variable performance (sensitivity and specificity) of these assays are to be expected given the differences in methodological and technical aspects (skills of the bronchoscopist, lavage technique and the BAL processing protocol), tuberculosis case definitions (culture confirmation alone vs a clinical definition for tuberculosis) and the populations studied.3 4 What both studies indicate, however, is that a BAL ELISpot would approximately double the yield of a rapid positive diagnosis over a smear alone. This additive value makes the test clinically promising. Further studies refining the assay and validating the cut-off points used in different settings are now required.


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  • Competing interests None.

  • Provenance and peer review Not commissioned; not externally peer reviewed.

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